Intracellular GTP Concentration Research Articles

Regulation of local GTP availability controls RAC1 activity and cell invasion

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells.

Abstract 1544: Cell penetrating single domain antibody (sdAb) SBT-100 binds KRAS and inhibits growth of human cancers with KRAS activating mutations

Abstract Background: Despite nearly fourth years of research, scientists have failed to develop a clinically viable therapy against KRAS, one of the deadliest families of cancer-causing proteins. Mutations in KRAS are prevalent amongst the top three most deadly cancer types in the United States: pancreatic (95%), colorectal (45%), and lung (35%). KRAS has been thought to be undruggable due to: 1) its intracellular location and lack of binding pockets for small molecules; 2) the high (pM) affinity of RAS for GTP precludes direct targeting of the nucleotide binding pocket; 3) high intracellular concentrations of GTP (uM) inhibits competition for the nucleotide-binding pocket by small molecules; and 4) possible toxicity. Mutations of KRAS result in it being perpetually turned on to propagate signal down the MAPK pathway. This results in constant production of P-ERK and plays an important role in malignant development. To overcome these challenges, Singh Biotechnology has developed SBT-100 a first in class & best in class novel sdAb that penetrates the cell membrane to bind KRAS to inhibit its GTPase activity. Methods: Human cancer cell lines were purchased from ATCC. BIAcore affinity studies were conducted by Precision Antibody. KRAS GTPase assay was purchased from Promega. Levels of P-ERK were determined using western blots. In vitro cell growth suppression was tested with MTT assay. Athymic nude mice for xenograft studies were purchased from Envigo. Results: SBT-100 binds KRAS with KD=10-9 and KRAS(G12D) with KD=10-7 as demonstrated by BIAcore affinity assay. Both SBT-100 and SBT-102 significantly inhibit KRAS GTPase activity in vitro and inhibition is comparable to polyclonal antibody to KRAS. Growth of MDA-MB-231 cells with KRAS(G13D) mutation and PANC-1 cells with KRAS(G12D) mutation are significantly decreased in the MTT assay when incubated with SBT-100. Additionally, same cell lines have significantly decreased P-ERK expression when cultured with SBT-100. Xenograft studies demonstrate significant growth suppression of MDA-MB-231 and PANC-1 when treated with SBT-100 in vivo. Conclusion: SBT-100, crosses the cell membrane, binds to KRAS intracellularly and its most common mutant with nanomolar affinity, inhibits KRAS GTPase activity, downregulates P-ERK signaling, and suppresses the growth of cancers cells in vitro and in vivo without showing any toxic effects. Citation Format: Sunanda Singh, Genoveva Murillo, Avani Singh, Samara Singh, Meenakshi Parihar, Anjali Singh, Rajendra Mehta, Ashutosh Parihar. Cell penetrating single domain antibody (sdAb) SBT-100 binds KRAS and inhibits growth of human cancers with KRAS activating mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1544.

Transcription Activity of IndividualrrnOperons inBacillus subtilisMutants Deficient in (p)ppGpp Synthetase Genes,relA,yjbM, andywaC

In Bacillus subtilis a null mutation of the relA gene, whose gene product is involved in the synthesis and/or hydrolysis of (p)ppGpp, causes a growth defect that can be suppressed by mutation(s) of yjbM and/or ywaC coding for small (p)ppGpp synthetases. All 35 suppressor mutations newly isolated were classified into two groups, either yjbM or ywaC, by mapping and sequencing their mutations, suggesting that there are no (p)ppGpp synthetases other than RelA, YjbM, and YwaC in B. subtilis. In order to understand better the relation between RelA and rRNA synthesis, we studied in the relA mutant the transcriptional regulation of seven rRNA operons (rrnO, -A, -J, -I, -E, -D, or -B) individually after integration of a promoter- and terminatorless cat gene. We identified the transcriptional start sites of each rrn operon (a G) and found that transcription of all rrn operons from their P1 promoters was drastically reduced in the relA mutant while this was almost completely restored in the relA yjbM ywaC triple mutant. Taken together with previous results showing that the intracellular GTP concentration was reduced in the relA mutant while it was restored in the triple mutant, it seems likely that continuous (p)ppGpp synthesis by YjbM and/or YwaC at a basal level causes a decrease in the amounts of intracellular GTP.

An alternative strategy for bacterial ribosome synthesis: Bacillus subtilis rRNA transcription regulation

As an approach to the study of rRNA synthesis in Gram-positive bacteria, we characterized the regulation of the Bacillus subtilis rrnB and rrnO rRNA promoters. We conclude that B. subtilis and Escherichia coli use different strategies to control rRNA synthesis. In contrast to E. coli, it appears that the initiating NTP for transcription from B. subtilis rRNA promoters is GTP, promoter strength is determined primarily by the core promoter (-10/-35 region), and changes in promoter activity always correlate with changes in the intracellular GTP concentration. rRNA promoters in B. subtilis appear to be regulated by changes in the initiating NTP pools, but in some growth transitions, changes in rRNA promoter activity are also dependent on relA, which codes for ppGpp synthetase. In contrast to the situation for E. coli where ppGpp decreases rRNA promoter activity by directly inhibiting RNA polymerase, it appears that ppGpp may not inhibit B. subtilis RNA polymerase directly. Rather, increases in the ppGpp concentration might reduce the available GTP pools, thereby modulating rRNA promoter activity indirectly.

Distinct Interactions of GTP, UTP, and CTP with GsProteins

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating G(s) protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support G(s) activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the beta(2)-adrenoreceptor (beta(2)AR) couples to G(s), and Sf9 insect cell membranes expressing beta(2)AR-Galpha(s) fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of beta(2)AR-G(s) coupling than native systems. Nucleoside 5'-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP > or = UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in beta(2)AR-Galpha(s) in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP > or = UTP > or = CTP > or = GTP. NTPs inhibited beta(2)AR-Galpha(s)-catalyzed [gamma-(32)P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Galpha(s) than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with G(s) proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach approximately 10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.

Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels.

CodY, a highly conserved protein in the low G + C, gram-positive bacteria, regulates the expression of many Bacillus subtilis genes that are induced as cells make the transition from rapid exponential growth to stationary phase and sporulation. This transition has been associated with a transient drop in the intracellular pool of GTP. Many stationary-phase genes are also induced during exponential-growth phase by treatment of cells with decoyinine, a GMP synthetase inhibitor. The effect of decoyinine on an early-stationary-phase gene is shown here to be mediated through CodY and to reflect a reduction in guanine nucleotide accumulation. CodY proved to bind GTP in vitro. Moreover, CodY-mediated repression of target promoters was dependent on a high concentration of GTP, comparable to that found in rapidly growing exponential-phase cells. Because a codY-null mutant was able to sporulate under conditions of nutrient excess, CodY also appears to be a critical factor that normally prevents sporulation under such conditions. Thus, B. subtilis CodY is a novel GTP-binding protein that senses the intracellular GTP concentration as an indicator of nutritional conditions and regulates the transcription of early-stationary-phase and sporulation genes, allowing the cell to adapt to nutrient limitation.

Involvement of IMP dehydrogenase activity in induction of sporulation of Bacillus cereus

IMP dehydrogenase activity of B. cereus increased parallel to cell growth in YE-EMM, where B. cereus did not sporulate. When B. cereus was cultured in a modified G medium, a sporulation medium, the activity reached the highest level at 6 hr and decreased thereafter. After induction of sporulation by nutritional shift down in 1/100 G medium, the enzyme activity decreased to about 5% compared with exponentially growing cells at 1 hr of resuspension. The sporulation rate of B. cereus was over 90% in the modified G medium and 1/100 G medium. Sporulation was strongly inhibited by mycophenolic acid at 1 mM, when the drug was added at 0 and 1 hr of resuspension in 1/100 G medium. Intracellular GTP concentration of B. cereus decreased to the lowest level about 1 hr of resuspension. Although GTP increased to about 50% of the exponentially growing cells at 2 hr of resuspension in control cells, the concentration did not increase in the presence of 1 mM mycophenolic acid.

Guanosine potentiates the antiproliferative effect of cytosine-beta-D-arabinofuranoside in melanoma cell lines.

Guanosine is shown to potentiate markedly the antiproliferative effect of cytosine-beta-D-arabinoside (ara-C) on B16 F10 mouse and SKMEL-28 human melanoma cell lines. Several metabolic consequences of the synergistic interaction between ara-C and guanosine on cell growth were determined in B16 F10 mouse melanoma cells. Treatment of the cells with guanosine for 24 hr resulted in an increase in the percentage of cells in the S phase of the cell cycle, a threefold increase in intracellular GTP concentration, and an increase in the incorporation of ara-C into acid-insoluble material and phosphorylated metabolites. These findings suggest that guanosine potentiates the growth-inhibitory effect of ara-C in B16 F10 melanoma cells by increasing the intracellular concentration of its active metabolites.

Mycophenolic acid simultaneously reduces intracellular GTP and tetrahydrobiopterin levels in neuro-2A cells.

Tetrahydrobiopterin (BH4) is involved in neural, immune and lipid functions as a natural cofactor for the aromatic amino acid hydroxylases1, the O-alkylglycerolipid cleavage enzyme2, and nitric oxide synthases3–5. In contrast to other coenzymes, BH4 is thought to be a regulator of these enzymes, because its intracellular concentration is within the range capable of affecting enzyme activities and is known to increase in response to the action of cytokines6–9 and in G/S boundary in rat thymocytes10, and to decrease with the differentiation of erythroid cells11,12. BH4 is synthesized from GTP by sequential actions of GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase. The biosynthesis of BH4 is mainly regulated at the step of GTP cyclohydrolase I, a ratelimiting enzyme whose activity is increased by a number of cytokines13–17. In addition to the probable regulation of its transcription10, we proposed that GTP cyclohydrolase I is regulated according to the availability of GTP, since we have observed cooperative binding of GTP to this enzyme18. To determine whether or not the level of intracellular GTP is within a range that can affect GTP cyclohydrolase I activity, we examined the effect of changes in the GTP level on the level of BH4 19; IMP dehydrogenase inhibitors, which inhibit the rate-limiting and committing step in de novo synthesis of GTP, were used to reduce the level of intracellular GTP, and guanine or guanosine was used to increase it. These experiments provided evidence that the intracellular GTP concentration in rat PC-12 pheochromocytoma cells and human IMR-32 neuroblastoma cells is the minimum required to elicit the maximal activity by GTP cyclohydrolase I. This supports the theory that GTP might regulate of the reaction catalyzed by GTP cyclohydrolase I19. In this study, we attempted to confirm and characterize the GTP-BH4 relationship using mouse Neuro-2a neuroblastoma cells.

Cell surface complexes (‘cortices’) isolated from Paramecium tetraurelia cells as a model system for analysing exocytosis in vitro in conjunction with microinjection studies

Cortex preparations isolated from Paramecium tetraurelia cells consist of surface with secretory organelles (trichocysts) still attached. In the absence of nucleotides, in media with a pCa of 5-5.5 and a pH of greater than or equal to 6.5, maximal exocytosis occurred when the Mg2+ concentration was lowered from 10 to 0.5 mM. ATP, as well as its non-hydrolysable analogues adenosine 5'-[gamma-thio]triphosphate (ATP[S]) and adenosine 5'[beta gamma-imido]triphosphate (App[NH]p), inhibited exocytosis at a concentration equivalent to that occurring in vivo (as determined by h.p.l.c.), but preincubation with ATP augmented the exocytotic response. GTP and its analogues only slightly stimulated exocytosis in vitro, but sensitivity to Ca2+ was increased significantly, in particular with GTP. These effects of nucleotides were rapidly reversible. Intracellular GTP concentrations (0.35 mM) would suffice for full activation with the pCai values assumed to occur in these cells during activation. On microinjection, ATP inhibited the secretagogue response in intact cells. Whereas microinjected GTP stimulated exocytosis (membrane fusion) without a secretagogue added, Gpp[NH]p remained without any effect; GTP[S] permanently abolished any triggered secretory response. Concomitantly, h.p.l.c. analysis of triggered and untriggered cells showed that GTP hydrolysis occurs immediately after synchronous (1 s) exocytosis in vivo. The precise site(s) of action of GTP during signal transduction in Paramecium cells remain to be determined.

Regulation of the phosphorylation state of Gi2 in intact rat hepatocytes

Hormonal stimulation and inhibition of intracellular signalling systems such as adenylate cyclase, phospholipases and ion channels require the activation of specific guanine nucleotide-binding G-proteins [ I ] . In addition to regulation by occupied hormone receptors, certain G-proteins may be partially activated, in the absence of extracellular signals, as a consequence of the high intracellular GTP concentrations ( 600 ,LAM). Thus, such G-proteins will exert a tonic stimulatory or inhibitory effect upon their target effector systems. G-proteins are also key sites for ‘cross-talk between the different signalling pathways. This can result in the phenomena of desensitization and of potentiation of specific signalling systems. Such ‘cross-talk’ represents an important mechanism for the control of cellular responsiveness and may be mediated by hormone-induced G-protein phosphorylation [2, 31. Using intact rat hepatocytes, we have examined hormonal regulation of phosphorylation of the G-proteins which control the activity of adenylate cyclase. Hepatocytes express both the G,2 and G,3 forms of the ‘Gi-family’ of G-proteins, but not G, 1. At least one of these G-proteins is believed to couple hormone receptors to the inhibition of adenylate cyclase. We have previously described the production, characterization and use of anti-peptide antisera which selectively recognize the a-subunits of various G-proteins [4]. These anti-peptide antisera were used to immunoprecipitate, selectively, either G,2 (antiserum AS 7), G,3 (antiserum 13B) or G, (antiserum CS1) and their subunits were resolved by polyacrylamide-gel electrophoresis. Using this immunoprecipitation procedure, we found that treatment of intact, 32P-labelled rat hepatocytes with glucagon ( 10 nM, 5 min), TH-glucagon (10 nM, 5 min), arginine-vasopressin (3 nM, S min), angiotensin II (3 nM, 5 min) and the horbol ester 12-0-tetradecanoyl phorbol 13acetate (TPAp( 10 n M , 15 min) elicited 2-2.5-fold maximal increases in the labelling of the a-subunit of immunoprecipitated G12.8-Bromo cyclic AMP (100 ,LAM, 15 min) and higher concentrations of glucagon (1 p ~ , 5 min) which act, at least in part, by activation of A-kinase caused 3.3-4-fold increases (Fig. 1). The effect of 8-bromo cyclic AMP was somewhat slower in onset (peak at 15 min of incubation) than the G12a phosphorylation induced by the protein kinase C activators (peak at 5 min). This may reflect ( u ) the time taken for permeation of this lipid-soluble cyclic AMP analogue to its site of action or (6) an indirect mode of action. The latter explanation is supported by the finding that G12a lacks a known consensus sequence for A-kinase-mediated phosphorylation [ 11. Phosphorylation of G12a was associated with loss of G, function as assessed by the ability of low concentrations ( 1 L

Growth inhibition of putrefactive anaerobe 3679 caused by stringent-type response induced by protonophoric activity of sorbic acid.

The inhibitory effects of potassium sorbate on the bioenergetics, phenylalanine uptake, protein synthesis, and certain aspects of cell regulation were examined in putrefactive anaerobe 3679. Undissociated sorbic acid appeared to act as a protonophore by lowering the intracellular pH and dissipating the proton motive force of the membrane. Sorbate inhibited the uptake of phenylalanine, decreased the rate of protein synthesis, and altered patterns of phosphorylated nucleotide accumulation, resulting in increased intracellular concentrations of GTP, ppGpp, and an unidentified compound (possibly pppGpp). The addition of a noninhibitory amount of tetracycline released the inhibition of growth by sorbate. Based on these results, we concluded that the inhibition of putrefactive anaerobe 3679 by sorbate resulted from a stringent-type regulatory response induced by the protonophoric activity of sorbic acid.

Initiation of meiosis and sporulation of Saccharomyces cerevisiae by sulfur or guanine deprivation

Homothallic Saccharomyces cerevisiae, growing exponentially in a synthetic acetate medium, could be initiated to undergo meiosis and subsequent sporulation by removal of sulfur from the medium or by partial purine deprivation of purine auxotrophs or, most efficiently, by guanine deprivation of a guanine auxotroph. In contrast, partial uracil deprivation of uracil auxotrophs did not cause sporulation. Under any of the above and other sporulation conditions, the intracellular concentrations of GTP and, usually at some time later, S-adenosylmethionine (SAM) decreased; the concentrations of the other nucleoside triphosphates decreased under some but increased under other sporulation conditions. The addition of 1 m m methionine or, more effectively, of SAM or the combination of adenine plus methionine greatly increased the intracellular concentration of SAM and reduced or prevented sporulation, even when GTP decreased. However, differentiation can be inhibited by an excess of many metabolites which do not specifically control the initiation process; in particular, SAM is known to inhibit yeast metabolism (e.g., transamination). Therefore, we cannot yet decide whether the deficiency of GTP or SAM (or related compounds) serves as a signal for the initiation of meiosis/sporulation.

Properties of beta-adrenoceptor sites in metabolizing and nonmetabolizing rat reticulocytes and in resealed reticulocyte ghosts.

Intact cells and resealed ghosts of a homogeneous reticulocyte population isolated from the blood of phenylhydrazine-treated rats bound β-adrenergic ligands reversibly, stereospecifically and with high affinity. Maximal specific binding capacity under control conditions (37° C), corresponded to 9.9 ± 0.8 fmol/μl cells (∼ 600 sites/cell) and was similar in intact cells and ghosts. Pretreatment with metabolic inhibitors decreased the density of binding sites in intact cells to 6.48 ± 1.1 fmol/μl cells, but had no effect in ghosts. Incubation at 1°C reduced specific binding in paired experiments by 68 and 44% in intact cells and ghosts, respectively. Rewarming to 37°C increased specific binding in cells and ghosts by 270 and 190%, respectively. A temperature shift from 1 to 37°C reduced the KD value for the antagonist (-)timolol from 8.6 ± 1.5 to 1.1 ± 0.3 nmol/l in intact cells, while no significant reduction in KD was observed with ghosts. Under all conditions the receptor population was homogeneous with respect to antagonist affinity but inhomogenous with respect to agonist affinity. Low affinity agonist binding sites predominated in native cells and in GTP- loaded resealed ghosts (apparent KD values 447 and 680 nmol/l, respectively). High affinity binding sites predominated in both preparations at 1°C (KD 29 and 14 nmol/l, respectively). β-Adrenoceptor sites in starved cells and ghosts at 37°C showed intermediate apparent KD values. GTP had no effect on antagonist affinity or on the density of β-adrenoceptors. The results suggest that intact metabolizing cells can regulate β-adrenoceptor density by mechanisms which are not shared by ghost cells. The fractional contribution of high and low affinity states of the receptor to the overall binding of agonists seemed to be determined largely by the intracellular GTP concentration.

Regulation of enzymes of purine metabolism in intact tumor cells

Studies have been made of the regulation of individual enzymes of purine ribonucleotide metabolism in intact Ehrlich ascites tumor cells incubated in vitro. Amidophosphoribosyltransferase, the first enzyme of the pathway of purine biosynthesis de novo, was not inhibited in cells that contained elevated concentrations of purine ribonucleotides, whereas phosphoribosyl pyrophosphate (PRPP) synthetase activity was inhibited under the same conditions. However, the rate of PRPP synthesis was found to be greater in the presence of purine bases than in their absence. The major factor responsible for inhibition of purine biosynthesis de novo when cells were incubated with purine bases was lowered concentrations of PRPP. Ribonucleotide synthesis from purine bases was limited by availability of PRPP, and was inhibited when cells contained elevated concentrations of purine nucleotides. The apparent activities of adenylate deaminase and inosinate dehydrogenase were inhibited by high intracellular concentrations of GTP, and the activities of guanylate synthetase and adenylosuccinate synthetase were limited by concentrations of glutamine and aspartate, respectively. When cells were incubated with deoxyglucose or dinitrophenol, rates of adenylate deamination and inosinate dephosphorylation increased greatly. However, these processes did not appear to be regulated by concentrations of ATP. It is concluded that endproduct inhibition of enzymes by purine ribonucleotides is not an important mode of regulation of purine metabolism in intact tumor cells.