Search

Purines are required for fundamental biological processes and alterations in their metabolism lead to severe genetic diseases associated with developmental defects whose etiology remains unclear. Here, we studied the developmental requirements for purine metabolism using the amphibian Xenopus laevis as a vertebrate model. We provide the first functional characterization of purine pathway genes and show that these genes are mainly expressed in nervous and muscular embryonic tissues. Morphants were generated to decipher the functions of these genes, with a focus on the adenylosuccinate lyase ( ADSL ), which is an enzyme required for both salvage and de novo purine pathways. adsl.L knockdown led to a severe reduction in the expression of the myogenic regulatory factors (MRFs: Myod1, Myf5 and Myogenin), thus resulting in defects in somite formation and, at later stages, the development and/or migration of both craniofacial and hypaxial muscle progenitors. The reduced expressions of hprt1.L and ppat , which are two genes specific to the salvage and de novo pathways, respectively, resulted in similar alterations. In conclusion, our data show for the first time that de novo and recycling purine pathways are essential for myogenesis and highlight new mechanisms in the regulation of MRF gene expression.

Purine homeostasis is ensured through a metabolic network widely conserved from prokaryotes to humans. Purines can either be synthesized de novo , reused, or produced by interconversion of extant metabolites using the so-called recycling pathway. Although thoroughly characterized in microorganisms, such as yeast or bacteria, little is known about regulation of the purine biosynthesis network in metazoans. In humans, several diseases are linked to purine metabolism through as yet poorly understood etiologies. Particularly, the deficiency in adenylosuccinate lyase (ADSL)-an enzyme involved both in the purine de novo and recycling pathways-causes severe muscular and neuronal symptoms. In order to address the mechanisms underlying this deficiency, we established Caenorhabditis elegans as a metazoan model organism to study purine metabolism, while focusing on ADSL. We show that the purine biosynthesis network is functionally conserved in C. elegans Moreover, adsl-1 (the gene encoding ADSL in C. elegans ) is required for developmental timing, germline stem cell maintenance and muscle integrity. Importantly, these traits are not affected when solely the de novo pathway is abolished, and we present evidence that germline maintenance is linked specifically to ADSL activity in the recycling pathway. Hence, our results allow developmental and tissue specific phenotypes to be ascribed to separable steps of the purine metabolic network in an animal model., (Copyright © 2019 by the Genetics Society of America.)

Metabolism is a highly integrated process resulting in energy and biomass production. While individual metabolic routes are well characterized, the mechanisms ensuring crosstalk between pathways are poorly described, although they are crucial for homeostasis. Here, we establish a co-regulation of purine and pyridine metabolism in response to external adenine through two separable mechanisms. First, adenine depletion promotes transcriptional upregulation of the de novo NAD + biosynthesis genes by a mechanism requiring the key-purine intermediates ZMP/SZMP and the Bas1/Pho2 transcription factors. Second, adenine supplementation favors the pyridine salvage route resulting in an ATP-dependent increase of intracellular NAD + . This control operates at the level of the nicotinic acid mononucleotide adenylyl-transferase Nma1 and can be bypassed by overexpressing this enzyme. Therefore, in yeast, pyridine metabolism is under the dual control of ZMP/SZMP and ATP, revealing a much wider regulatory role for these intermediate metabolites in an integrated biosynthesis network., Competing Interests: BP, JC, CS, BD No competing interests declared, (© 2019, Pinson et al.)

Purine nucleotides are involved in a multitude of cellular processes, and the dysfunction of purine metabolism has drastic physiological and pathological consequences. Accordingly, several genetic disorders associated with defective purine metabolism have been reported. The etiology of these diseases is poorly understood and simple model organisms, such as yeast, have proved valuable to provide a more comprehensive view of the metabolic consequences caused by the identified mutations. In this review, we present results obtained with the yeast Saccharomyces cerevisiae to exemplify how a eukaryotic unicellular organism can offer highly relevant information for identifying the molecular basis of complex human diseases. Overall, purine metabolism illustrates a remarkable conservation of genes, functions and phenotypes between humans and yeast.

Previous genetic analyses showed phenotypic interactions between 5-amino-4-imidazole carboxamide ribonucleotide 5'-phosphate (AICAR) produced from the purine and histidine pathways and methionine biosynthesis. Here, we revisited the effect of AICAR on methionine requirement due to AICAR accumulation in the presence of the fau1 mutation invalidating folinic acid remobilization. We found that this methionine auxotrophy could be suppressed by overexpression of the methionine synthase Met6 or by deletion of the serine hydroxymethyltransferase gene SHM2. We propose that in a fau1 background, AICAR, by stimulating the transcriptional expression of SHM2, leads to a folinic acid accumulation inhibiting methionine synthesis by Met6. In addition, we uncovered a new methionine auxotrophy for the ade3 bas1 double mutant that can be rescued by overexpressing the SHM2 gene. We propose that methionine auxotrophy in this mutant is the result of a competition for 5,10-methylenetetrahydrofolate between methionine and deoxythymidine monophosphate synthesis. Altogether, our data show intricate genetic interactions between one-carbon units, purine and methionine metabolism through fine-tuning of serine hydroxymethyltransferase by AICAR and the transcription factor Bas1.

5-Amino-4-imidazolecarboxamide ribonucleotide 5'-phosphate (AICAR) is a monophosphate metabolic intermediate of the de novo purine synthesis pathway that has highly promising metabolic and antiproliferative properties. Yeast mutants unable to metabolize AICAR are auxotroph for histidine. A screening for suppressors of this phenotype identified recessive and dominant mutants that result in lowering the intracellular AICAR concentration. The recessive mutants affect the adenosine kinase, which is shown here to catalyze the phosphorylation of AICAR riboside in yeast. The dominant mutants strongly enhance the capacity of the alkaline phosphatase Pho13 to dephosphorylate 5-amino-4-imidazole N-succinocarboxamide ribonucleotide 5'-phosphate(SAICAR) into its non-toxic riboside form. By combining these mutants with transcriptomics and metabolomics analyses, we establish that in yeast responses to AICAR and SAICAR are clearly linked to the concentration of the monophosphate forms, whereas the derived nucleoside moieties have no effect even at high intracellular concentration. Finally, we show that AICAR/SAICAR concentrations vary under physiological conditions known to modulate transcription of the purine and phosphate pathway genes.

Cells use strategic metabolites to sense the metabolome and accordingly modulate gene expression. Here, we show that the purine and phosphate pathways are positively regulated by the metabolic intermediate AICAR (5'-phosphoribosyl-5-amino-4-imidazole carboxamide). The transcription factor Pho2p is required for up-regulation of all AICAR-responsive genes. Accordingly, the binding of Pho2p to purine and phosphate pathway gene promoters is enhanced upon AICAR accumulation. In vitro, AICAR binds both Pho2p and Pho4p transcription factors and stimulates the interaction between Pho2p and either Bas1p or Pho4p in vivo. In contrast, SAICAR (succinyl-AICAR) only affects Pho2p-Bas1p interaction and specifically up-regulates purine regulon genes. Together, our data show that Bas1p and Pho4p compete for Pho2p binding, hence leading to the concerted regulation of cellular nucleotide synthesis and phosphate consumption.

Adenylate kinase (Adk1p) is a pivotal enzyme in both energetic and adenylic nucleotide metabolisms. In this paper, using a transcriptomic analysis, we show that the lack of Adk1p strongly induced expression of the PHO and ADE genes involved in phosphate utilization and AMP de novo biosynthesis respectively. Isolation and characterization of adk1 point mutants affecting PHO5 expression revealed that all these mutations also severely affected Adk1p catalytic activity, as well as PHO84 and ADE1 transcription. Furthermore, overexpression of distantly related enzymes such as human adenylate kinase or yeast UMP kinase was sufficient to restore regulation. These results demonstrate that adenylate kinase catalytic activity is critical for proper regulation of the PHO and ADE pathways. We also establish that adk1 deletion and purine limitation have similar effects on both adenylic nucleotide pool and PHO84 or ADE17 expression. Finally, we show that, in the adk1 mutant, upregulation of ADE1 depends on synthesis of the previously described effector(s) (S)AICAR ((N-succinyl)-5-aminoimidazol-4-carboxamide ribotide), while upregulation of PHO84 necessitates the Spl2p positive regulator. This work reveals that adenylic nucleotide availability is a key signal used by yeast to co-ordinate phosphate utilization and purine synthesis.

Because some metabolic intermediates are involved in more than one pathway, crosstalk between pathways is crucial to maintaining homeostasis. AMP and histidine biosynthesis pathways are coregulated at the transcriptional level in response to adenine availability. 5'-Phosphoribosyl-4-carboxamide-5-aminoimidazole (AICAR), a metabolic intermediate at the crossroads between these two pathways, is shown here to be critical for activation of the transcriptional response in the absence of adenine. In this study, we show that both AMP and histidine pathways significantly contribute to AICAR synthesis. Furthermore, we show that upregulation of the histidine pathway clearly interferes with regulation of the AMP pathway, thus providing an explanation for the regulatory crosstalk between these pathways. Finally, we revisit the histidine auxotrophy of ade3 or ade16 ade17 mutants. Interestingly, overexpression of PMU1, encoding a potential phosphomutase, partially suppresses the histidine requirement of an ade3 ade16 ade17 triple mutant, most probably by reducing the level of AICAR in this mutant. Together our data clearly establish that AICAR is not just a metabolic intermediate but also acts as a true regulatory molecule.

Glutamine, glycine and 10-formyl tetrahydrofolate are consumed during de novo purine biosynthesis. We have found that, in Saccharomyces cerevisiae, synthesis of these cosubstrates is coregulated with synthesis of enzymes of the purine biosynthetic pathway. Analysis of three genes required for synthesis of glutamine, glycine and 10-formyl tetrahydrofolate (GLN1, SHM2 and MTD1, respectively) shows that their expression is repressed by adenine and requires the transcription factors Baslp and Bas2p. Northern analysis reveals that regulation of SHM2 and MTD1 expression by adenine takes place at the transcriptional level. We also show that Bas1p and Bas2p bind in vitro to the promoters of the SHM2 and MTD1 genes, and that mutations in the consensus Bas1p binding sequences strongly affect expression of these genes in vivo. Finally, we have found that a SHM2-lacZ fusion is expressed at a significantly higher level in a bas2-2 disrupted strain than in bas1-2 or bas1-2 bas2-2 mutant strains. The BAS1-dependent, BAS2-independent expression of SHM2-lacZ suggests that, in the absence of Bas2p, Bas1p can interact with another protein partner to activate SHM2 expression.

We have found cross-pathway regulation between purine and histidine biosynthesis in yeast. The transcription factors BAS1 and BAS2/PHO2, which are also regulators of the histidine pathway, participate in the regulation of the purine biosynthetic pathway. Analysis of four genes of the purine pathway (ADE1, ADE2, ADE5,7, and ADE8) shows that their expression is repressed by adenine. The maximal basal and induced expression of these purine genes requires the presence of both BAS1 and BAS2. The factor BAS1 has been shown to bind at a site containing the TGACTC hexanucleotide motif in the ADE2 and ADE5,7 promoters. This motif is required for both basal and induced activation of the ADE2 gene by BAS1 and BAS2.