Regulation Of Purine Biosynthesis Research Articles

Abstract A10: Exploring the interplay between the purinosome, a multienzyme, purine biosynthetic machine, and the Rheb-mTORC1 signaling axis

Abstract Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. We have previously demonstrated that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme, GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, in part due to a reduction in intracellular guanine nucleotides. Purines are synthesized intracellularly in dynamic metabolic structures known as the purinosome. Our emerging data suggest that Rheb associates with the purinosome and treatment with AG2037 disrupts this interaction. We hypothesize that Rheb senses the availability of purines at the site of the purinosome and communicates purine sufficiency to activate mTORC1. The results of this project will increase our understanding of the regulation of purine biosynthesis and mTORC1 activation, two key processes in cell proliferation and cancer. Citation Format: Natasha Emmanuel, Elyssa Bernfeld, Shoba Ragunathan, Fang Wang, Qin Shan, Andreas Giannakou, Nanni Huser, Guixian Jin, Jeremy Myers, Keziban Unsal-Kacmaz, Robert T. Abraham. Exploring the interplay between the purinosome, a multienzyme, purine biosynthetic machine, and the Rheb-mTORC1 signaling axis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A10.

The Stringent Response Contributes to Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection Through the Purine Biosynthetic Pathway.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinical-therapeutic challenge. Of particular concern is antibiotic treatment failure in infections caused by MRSA that are "susceptible" to antibiotic in vitro. In the current study, we investigate specific purine biosynthetic pathways and stringent response mechanism(s) related to this life-threatening syndrome using genetic matched persistent and resolving MRSA clinical bacteremia isolates (PB and RB, respectively), and isogenic MRSA strain sets. We demonstrate that PB isolates (vs RB isolates) have significantly higher (p)ppGpp production, phenol-soluble-modulin expression, polymorphonuclear leukocyte lysis and survival, fibronectin/endothelial cell (EC) adherence, and EC damage. Importantly, an isogenic strain set, including JE2 parental, relP-mutant and relP-complemented strains, translated the above findings into significant outcome differences in an experimental endocarditis model. These observations indicate a significant regulation of purine biosynthesis on stringent response, and suggest the existence of a previously unknown adaptive genetic mechanism in persistent MRSA infection.

DRES-09. REGULATORY EFFECTS OF THE CILIARY GTPASE ARL13B ON PURINE METABOLISM IN GBM

Abstract Glioblastoma (GBM) is the most common form of adult primary brain cancer. Despite an aggressive treatment regimen – surgical resection, irradiation, and temozolomide (TMZ) chemotherapy – patients’ prognosis is still grim. TMZ acts by methylating purines, specifically at the O6 and N7 positions of guanine, to induce cytotoxic DNA double-strand breaks. We thus wanted to explore how purine metabolism may contribute to TMZ-resistance. In mammalian cells, purine nucleotides can be recycled by the salvage pathway or generated via de novo synthesis. The salvage pathway is energetically inexpensive relative to de novo thus, highly proliferative GBM cells preferentially utilize the salvage pathway. We have shown that salvage synthesis is reduced in response to TMZ (p-value=0.0021), hinting that the cells may utilize de novo to evade therapy induced alkylation of purines. Using immunoprecipitation-mass spectroscopy analysis, we found a novel interaction between the ciliary GTPase ARL13B and IMPDH2, the rate-limiting enzyme in de novo synthesis. We have shown that this interaction, occurring at the C-terminal domain of ARL13B, plays a significant role in the regulation of purine biosynthesis as abolishing it through ARL13B knockdown reduced flux through de novo (p-value< 0.0001) synthesis as measured by the specific activity of IMPDH2. Further, the lentiviral-mediated rescue of ARL13B brings IMPDH2 activity back to basal levels (p< 0.0001). Given its canonical function as a GTPase, we hypothesize that ARL13B acts as a novel regulator of de novo synthesis by sequestering GDP, allowing IMPDH2 to sense and respond to the cytosolic levels of guanine nucleotides. Without ARL13B the de novo pathway is halted, forcing the cells to rely on salvage to replenish nucleotide pools. Reliance on this pathway in the presence of TMZ causes cells to incorporate damaged nucleotides as a result of the drug’s alkylating action leading to the increased therapeutic efficacy of TMZ.

Abstract B13: Akt-independent PDK1 signaling in regulation of purine biosynthesis

Abstract Molecular association of cancer cell metastasis with signaling pathways has been explicated so as to aid in the development of new prognostic models for better cancer therapies. However, those metastatic signaling pathways are barely explored to take account of the functions of enzymes involved in cellular metabolism. Particularly, the metabolic enzymes in de novo purine biosynthesis have been overlooked for their potential roles in cancer cell metastasis even though they have been successfully validated anti-cancer drug targets. Meanwhile, several lines of recent discoveries on de novo purine biosynthesis suggest that the spatiotemporal assembly of purine biosynthetic enzymes, the purinosome, is under controls of signaling pathways in cancer cells. The results of the inquiry reveal an unanticipated mechanism of action of 3-phosphoinositide-dependent protein kinase 1 (PDK1) signaling pathways in regulation of purine biosynthesis in an Akt-independent manner. Considering the biological action of the Akt-independent PDK1 signaling in anchorage-independent growth of breast cancer cells, we hypothesize that purinosome assembly and disassembly are regulated by Akt-independent PDK1 signaling pathways during breast cancer cell migration and invasion. Collectively, the research coupling two druggable anti-cancer targets will reveal the importance of the heretofore unrecognized molecular interaction between the Akt-independent PDK1 signaling and the purinosome, leading to therapeutic intervention in the management of breast cancer metastasis. Citation Format: Songon An. Akt-independent PDK1 signaling in regulation of purine biosynthesis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B13.

Eaf1 Links the NuA4 Histone Acetyltransferase Complex to Htz1 Incorporation and Regulation of Purine Biosynthesis.

Proper modulation of promoter chromatin architecture is crucial for gene regulation in order to precisely and efficiently orchestrate various cellular activities. Previous studies have identified the stimulatory effect of the histone-modifying complex NuA4 on the incorporation of the histone variant H2A.Z (Htz1) at the PHO5 promoter (A. Auger, L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Côté, Mol Cell Biol 28:2257-2270, 2008, http://dx.doi.org/10.1128/MCB.01755-07). In vitro studies with a reconstituted system also indicated an intriguing cross talk between NuA4 and the H2A.Z-loading complex, SWR-C (M. Altaf, A. Auger, J. Monnet-Saksouk, J. Brodeur, S. Piquet, M. Cramet, N. Bouchard, N. Lacoste, R. T. Utley, L. Gaudreau, J. Côté, J Biol Chem 285:15966-15977, 2010, http://dx.doi.org/10.1074/jbc.M110.117069). In this work, we investigated the role of the NuA4 scaffold subunit Eaf1 in global gene expression and genome-wide incorporation of Htz1. We found that loss of Eaf1 affects Htz1 levels mostly at the promoters that are normally highly enriched in the histone variant. Analysis of eaf1 mutant cells by expression array unveiled a relationship between NuA4 and the gene network implicated in the purine biosynthesis pathway, as EAF1 deletion cripples induction of several ADE genes. NuA4 directly interacts with Bas1 activation domain, a key transcription factor of adenine genes. Chromatin immunoprecipitation (ChIP) experiments demonstrate that nucleosomes on the inactive ADE17 promoter are acetylated already by NuA4 and enriched in Htz1. Upon derepression, these poised nucleosomes respond rapidly to activate ADE gene expression in a mechanism likely reminiscent of the PHO5 promoter, leading to nucleosome disassembly. These detailed molecular events depict a specific case of cross talk between NuA4-dependent acetylation and incorporation of histone variant Htz1, presetting the chromatin structure over ADE promoters for subsequent chromatin remodeling and activated transcription.

Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB

Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

Modifiers ofPrat, a de novo purine synthesis gene, inDrosophila melanogaster

Drosophila melanogaster was used to identify genes with a potential role in genetic regulation of purine biosynthesis. In this study we examine two dominant genetic modifiers of the essential gene Prat, which encodes amidophosphoribosyltransferase (EC 2.4.2.14). We found that Mod(Prat:bw)3-1 enhances Prat expression only in female heads, whereas Mod(Prat:bw)3-5 suppresses Prat in all stages and tissues examined for both sexes. For Mod-3-5, gene expression microarrays were used to identify other genes that are affected by the modifier. Three mapping approaches were used to localize these modifiers. Deficiency and meiotic mapping showed that the complex lethal complementation group previously associated with Mod-3-1 and Mod-3-5 is actually due to shared second-site lethal mutations. Using male recombination mapping, Mod-3-1 was localized to a 21 kilobase region containing nine genes, and Mod-3-5 was localized to a 53 kilobase region containing eight genes.

Transcriptome and Functional Analysis of the Eukaryotic-Type Serine/Threonine Kinase PknB inStaphylococcus aureus

The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn(2+) as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.

Regulation of purine biosynthesis by a eukaryotic‐type kinase in Streptococcus agalactiae

Group B streptococci (GBS) are the principal causal agents of human neonatal pneumonia, sepsis and meningitis. We had previously described the existence of a eukaryotic-type serine/threonine kinase (Stk1) and phosphatase (Stp1) in GBS that regulate growth and virulence of the pathogen. Our previous results also demonstrated that these enzymes reversibly phosphorylated an inorganic pyrophosphatase. To understand the role of these eukaryotic-type enzymes on growth of GBS, we assessed the stk1-mutants for auxotrophic requirements. In this report, we describe that in the absence of the kinase (Stk1), GBS are attenuated for de novo purine biosynthesis and are consequently growth arrested. During growth in media lacking purines, the intracellular G nucleotide pools (GTP, GDP and GMP) are significantly reduced in the Stk1-deficient strains, while levels of A nucleotides (ATP, ADP and AMP) are marginally increased when compared with the isogenic wild-type strain. We provide evidence that the reduced pools of G nucleotides result from altered activity of the IMP utilizing enzymes, adenylosuccinate synthetase (PurA) and IMP dehydrogenase (GuaB) in these strains. We also demonstrate that Stk1 and Stp1 reversibly phosphorylate and consequently regulate PurA activity in GBS. Collectively, these data indicate the novel role of eukaryotic-type kinases in regulation of metabolic processes such as purine biosynthesis.

Ureide biosynthesis in legume nodules.

In tropical legumes like Glycine, Phaseolus and Vigna sp., ammonia as direct product of symbiotic nitrogen fixation is converted to ureides (allantoin and allantoic acid) and they were translocated to the shoots as nitrogen source. In the xylem sap of soybean in reproductive phase the ureides reached to 60-75% of soluble nitrogen. In nodules infected cells (plastid and mitochondria) and uninfected cells (peroxisome) shares de novo purine biosynthesis and urate oxidation to produce ureides respectively. Current research revealed unique feathers on this symbiotic metabolism, especially on regulation of purine biosynthesis, uricase gene expression and feedback inhibition of ureides to nitrogen fixing activity.

Chemiluminescent assays in the study of purine metabolism.

The discovery that in cells from patients with hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficiency purine overproduction is associated with an elevated intracellular concentration of phosphoribosylpyro-phosphate (PRPP) (1) has led to a reevaluation of the importance of this compound in the regulation of purine biosynthesis de novo. The work which flowed from this discovery led to the identification of an inherited super-activity of PRPP synthetase in over a dozen families in which affected individuals showed gout and/or uric acid overproduction (2). However to date HGPRT deficiency and increased activity of PRPP synthetase are the only two well documented enzyme defects of purine synthesis and reutilization identified among hyperuricemic patients. These genetic aberrations account for less 10% of all hyperuricemic patients who overproduce uric acid.

The hormonal regulation of purine biosynthesis: control of the inosinic acid branch point.

We previously studied the behavior of “ de novo ” purine biosynthesis and of RNA metabolism in the levator ani muscle (LAM) of castrated rats, demostrating that 1) “ de novo ” purine biosynthesis decreases in the LAM of the rat after castration, as shown by a diminishing incorporation of 14C-formate in all bases; 2) the ratio G/A ( = counts per min in guanine counts per min in adenine ), an index of IMP branched point regulation 2, is unchanged under such conditions; 3) since only the RNA content decrea ses, but not its specific activity, we concluded that the formation of new molecules of RNA remains the same, while the RNA breakdown is accelerated during the atrophy of the LAM which follows castration. Now we have stu died the behavior of purine biosynthesis “ de novo ” in the LAM of adult male Wistar rats, before, after castration and after testosterone administration.

Regulation of purine biosynthesis in G1 phase‐arrested mammalian cells

The effects of G1 phase growth arrest on purine biosynthesis were studied in cultured S49 T lymphoma cells. Incubations of wildtype S49 cells for 18 hr with dibutyryl cyclic AMP or forskolin, two agents which induced G1 arrest, reduced the rates of purine biosynthesis by 95%. Time course and concentration dependence studies indicated that the decrease in rates of purine biosynthesis correlated with the extent of G1 phase arrest. Similar studies with somatic cell mutants deficient in some component of cyclic AMP action or metabolism indicated that the depression in purine synthetic rates required G1 arrest and did not result from cell death. Rates of RNA and DNA synthesis were also markedly diminished in the growth arrested cells. Measurements of purine rates in the presence of azaserine indicated that the block in purine biosynthesis was prior to the formation of phosphoribosylformylglycinamide. Additionally, the activities of adenylosuccinate synthetase and IMP dehydrogenase were diminished in G1 arrested cells. The levels of all controlling enzymes, substrates, and cofactors, however, were not diminished in G1 arrested cells. Despite diminished rates of purine biosynthesis, the amounts of intracellular nucleotides in G1 cells were equivalent to those in exponentially growing cells. However, the concentrations of intracellular nucleotides were 30-50% higher in the growth arrested cells. These results suggested that perturbations in the consumption of nucleotides via inhibition of nucleic acid synthesis have profound effects on the purine pathway and indicated the importance of feedback inhibition by nucleotides in the regulation of purine synthesis in situ.

Plasma follicle-stimulating hormone, luteinizing hormone, and sex hormones in patients with gout.

Plasma levels of follicle-stimulating hormone, luteinizing hormone, testosterone, progesterone, 17 beta-estradiol, and cortisol were examined in normal subjects and gout patients under baseline conditions and after clomiphene stimulation. A significant decrease in follicle-stimulating hormone, luteinizing hormone, and 17 beta-estradiol was observed both in male and female gout patients; in the same patients, the plasma testosterone: 17 beta-estradiol ratio was found to be significantly higher than in control subjects. The changes observed suggest a possible role played by 17 beta-estradiol in the regulation of purine biosynthesis and uric acid metabolism.

Regulatory studies on purine biosynthesis in the chick.

Uricotelic animals use synthesis of the purine ring system as the pathway for excretion of excess nitrogen. This results in a high level of de novo purine synthesis (1). When birds are subjected to stress situations that lead to higher nitrogen excretion, the rate of purine synthesis is increased (2,3) and changes in levels of adenylosuccinate lyase, xanthine dehydrogenase and amidophosphoribosyltransferase occur (2,4). Livers from chickens treated with β-estradiol can provide a model system for a rapidly growing, normal cell. Treatment with this hormone causes a rapid increase in liver weight and in protein, RNA and DNA content in preparation for the production of egg proteins (5). Weber and coworkers (6–8) have shown that the level of certain purine biosynthetic enzymes is increased in regenerating rat liver, another system that is used as a model for normal, rapid growth. This is also true in certain rat liver hepatomas (6–8). The chick liver can therefore be used as a model for study of regulation of purine biosynthesis under conditions where requirements for nucleotides vary.

The hormonal regulation of purine biosynthesis.

The hormonal regulation of purine biosynthesis is not well known and there are more indications taken from clinical data then based on experimental ground.

Regulation of purine biosynthesis and interconversion in the chick.

In ureotelic species, such as the rat, adaptive changes in metabolic flux and enzyme levels occur in the purine metabolic pathway when cells are rapidly growing. This is observed in both regenerating liver and in malignant tissues. The enzymes P-Rib-PP amidotransferase and IMP dehydrogenase increase in activity in both situations. The level of purine biosynthesis is much higher in uricotelic species, such as the chick, when compared to ureotelic animals. By treating immature roosters with the hormone beta-estradiol, it is possible to induce rapid liver growth, allowing comparison of the regulation of purine biosynthesis and interconversion in high metabolic rate cells with different roles for purine metabolism. The tissue activities of P-Rib-PP amidotransferase, xanthine dehydrogenase, adenylosuccinate synthetase and lyase, AMP deaminase, IMP dehydrogenase, and GMP synthetase did not rise in livers from estradiol-treated chicks, as compared to controls. However, the rate of de novo purine synthesis triples and the intracellular level of P-Rib-PP doubles within 24 h of treatment. The biosynthesis of GMP is elevated at 12 and 24 h, but the levels of soluble nucleotide pools do not change. These data indicate that regulation of the de novo purine pathway in uricotelic species in a high metabolic situation is at the level of substrate availability (P-Rib-PP) and not due to changes in enzyme level or to feedback inhibition.

The potentiation of adenine toxicity to Chinese hamster cells by coformycin: suppression in mutants with altered regulation of purine biosynthesis or increased adenylate-deaminase activity.

When added to medium containing coformycin (2 microM or above), adenine is lethal to Chinese hamster fibroblasts at the concentration inhibiting de novo purine biosynthesis (Debatisse and Buttin, '77b). Rescue by hypoxanthine suggested that cells die of IMP starvation when the analog can turn off deamination of both adenosine and adenylate. As predicted from this hypothesis, two classes of variants resistant to the mixture of coformycin + adenine have been isolated: Class 1 variants have altered control of de novo IMP biosynthesis; they fall into two subclasses on the basis of their resistance to adenosine. Class 2 variants have a 6-10-fold increased level of AMP-deaminase (E.C.: 3.5.4.6); their growth in the selective medium is temperature-dependent, a property accounted for by the observation that cell growth in the presence of coformycin imposes a gradual thermodependent decay of specific AMP-deaminase activity in both wild-type and variant lines. This control by coformycin of AMP-deaminase activity is unaltered in mutants deficient in the four activities of adenosine-kinase. APRT, HGPRT and deoxycytidine-kinase. Most of the resistant variants are unstable and exhibit either increased or reduced resistance, depending on prolonged growth in selective or normal medium.

Regulation of purine biosynthesis in cultured Drosophila melanogaster cells : II/relationships between hypoxanthine-guanine-phosphoribosyl transferase and 5′-nucleotidase

All in vitro cell lines from Drosophila melanogaster do not possess measurable hypoxanthine - guanine - phosphoribosyltransferase (HGPRT) and 5'-nucleotidase activities. Nevertheless these enzymatic activities could be detected by the modification of culture conditions or treatment of crude extracts from Drosophila cells (1). A Drosophila melanogaster hemocyte line enabled demonstration of a relationship between the activities of HGPRT and 5'-nucleotidase. Only the addition of azaserine to the culture medium allows detection of a measurable amount of HGPRT and 5'-nucleotidase in cells devoid of endogenous synthesis of purine nucleotides. This action of azaserine suggests that the simultaneous expression of both enzymes is first directed by the suppression of the 5'-nucleotidase inhibition by pyrimidine nucleotides, and later on by the 5'-nucleotidase activity hydrolyzing the mononucleotides which inhibited the HGPRT, resulting its expression. This regulation system is confirmed by the simultaneous recovery of HGPRT and 5'-nucleotidase activities after heating cell cultures or cell extracts. Under such experimental conditions, 5'-nucleotidase is desensitized towards its negative effectors, while HGPRT regulation remains unchanged.

A mutant of CHO-K1 cells deficient in two nonsequential steps of de novo purine biosynthesis

An unusual new purine-requiring mutant, Ade −P AB, of Chinese hamster ovary cells (CHO-K1) is described. Ade −P AB will grow in medium supplemented with hypoxanthine, adenine, or aminoimidazole carboxamide. Ade −P AB fails to show genetic complementation with either Ade −A, defective in amidophosphoribosyltransferase (E.C. 2.4.2.14), or Ade −B, defective in phosphoribosylformylglycinamidine FGAM) synthetase (E.C. 6.3.5.3.), but will complement all five of our other hypoxanthine-requiring Ade − complementation groups. Analysis of purine synthesis in wild-type, mutant, and revertant cells and analysis of relevant enzyme activities in cell-free extracts prepared from these cells demonstrates that Ade −P AB is similar to Ade −B in that it has lost FGAM synthetase activity, and is similar to Ade −A in that it has lost glutamine-dependent amidophosphoribosyltransferase activity. Unlike Ade −A, however, Ade −P AB retains the ability to synthesize phosphoribosylamine (PRA), the product of the amidophosphoribosyltransferase reaction, if NH 4Cl is substituted for glutamine as the nitrogen donor. Moreover, partial revertants of Ade −P AB can apparently synthesize sufficient purines for growth using the NH 4Cl-dependent reaction. The available evidence indicates that neither a double mutation nor a deletion is probable in Ade −P AB. We discuss the relevance of these observations for our understanding of both the regulation of purine biosynthesis in mammalian cells and the structural organization of the enzymes defective in Ade −P AB and the genes coding for these enzymes.