A journey into the regulatory secrets of the de novo purine nucleotide biosynthesis
Ontology highlight
ABSTRACT:
SUBMITTER: Ayoub N
PROVIDER: S-EPMC10912719 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The focus of this review is the human de novo purine biosynthetic pathway. The pathway enzymes are enumerated, as well as the reactions they catalyze and their physical properties. Early literature evidence suggested that they might assemble into a multi-enzyme complex called a metabolon. The finding that fluorescently-tagged chimeras of the pathway enzymes form discrete puncta, now called purinosomes, is further elaborated in this review to include: a discussion of their assembly; the role of ancillary proteins; their locus at the microtubule/mitochondria interface; the elucidation that at endogenous levels, purinosomes function to channel intermediates from phosphoribosyl pyrophosphate to AMP and GMP; and the evidence for the purinosomes to exist as a protein condensate. The review concludes with a consideration of probable signaling pathways that might promote the assembly and disassembly of the purinosome, in particular the identification of candidate kinases given the extensive phosphorylation of the enzymes. These collective findings substantiate our current view of the de novo purine biosynthetic metabolon whose properties will be representative of how other metabolic pathways might be organized for their function.
| S-EPMC7869020 | biostudies-literature
Project description:A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C-, 15N-enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose, and CO2 as stoichiometric precursors in labeled form. Using this approach, U-13C- GTP, U-13C, 15N- GTP, 13C 2,8- ATP, and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region RNA containing 13C 2,8-adenosine and 15N 1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected, expanding the applicability of enzymatic synthesis.
| S-EPMC2746247 | biostudies-literature
Project description:Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, and autistic features. The pathogenetic mechanism is unknown for these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.
| S-EPMC4296673 | biostudies-literature
Project description:Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.
| S-EPMC6287904 | biostudies-literature
Project description:Purine biosynthesis by the 'de novo' pathway was demonstrated in isolated rat extensor digitorum longus muscle with [1-14C]glycine, [3-14C]serine and sodium [14C]formate as nucleotide precursors. Evidence is presented which suggests that the source of glycine and serine for purine biosynthesis is extracellular rather than intracellular. The relative incorporation rates of the three precursors were formate greater than glycine greater than serine. Over 85% of the label from formate and glycine was recovered in the adenine nucleotides, principally ATP. Azaserine markedly inhibited purine biosynthesis from both formate and glycine. Cycloserine inhibited synthesis from serine, but not from formate. Adenine, hypoxanthine and adenosine markedly inhibited purine synthesis from sodium [14C]formate.
| S-EPMC1152552 | biostudies-other
Project description:Dynamic partitioning of de novo purine biosynthetic enzymes into multienzyme compartments, purinosomes, has been associated with increased flux of de novo purine biosynthesis in human cells. However, we do not know of a mechanism by which de novo purine biosynthesis would be downregulated in cells. We have investigated the functional role of AMP-activated protein kinase (AMPK) in the regulation of de novo purine biosynthesis because of its regulatory action on lipid and carbohydrate biosynthetic pathways. Using pharmacological AMPK activators, we have monitored subcellular localizations of six pathway enzymes tagged with green fluorescent proteins under time-lapse fluorescence single-cell microscopy. We revealed that only one out of six pathway enzymes, formylglycinamidine ribonucleotide synthase (FGAMS), formed spatially distinct cytoplasmic granules after treatment with AMPK activators, indicating the formation of single-enzyme self-assemblies. In addition, subsequent biophysical studies using fluorescence recovery after photobleaching showed that the diffusion kinetics of FGAMS were slower when it localized inside the self-assemblies than within the purinosomes. Importantly, high-performance liquid chromatographic studies revealed that the formation of AMPK-promoted FGAMS self-assembly caused the reduction of purine metabolites in HeLa cells, indicating the downregulation of de novo purine biosynthesis. Collectively, we demonstrate here that the spatial sequestration of FGAMS by AMPK is a mechanism by which de novo purine biosynthesis is downregulated in human cells.
| S-EPMC5675104 | biostudies-literature
Project description:Nucleotide synthesis is essential to proliferating cells, but the preferred precursors for de novo biosynthesis are not defined in human cancer tissues. We have employed multiplexed stable isotope-resolved metabolomics to track the metabolism of [13C6]glucose, D2-glycine, [13C2]glycine, and D3-serine into purine nucleotides in freshly resected cancerous and matched noncancerous lung tissues from nonsmall cell lung cancer (NSCLC) patients, and we compared the metabolism with established NSCLC PC9 and A549 cell lines in vitro Surprisingly, [13C6]glucose was the best carbon source for purine synthesis in human NSCLC tissues, in contrast to the noncancerous lung tissues from the same patient, which showed lower mitotic indices and MYC expression. We also observed that D3-Ser was preferentially incorporated into purine rings over D2-glycine in both tissues and cell lines. MYC suppression attenuated [13C6]glucose, D3-serine, and [13C2]glycine incorporation into purines and reduced proliferation in PC9 but not in A549 cells. Using detailed kinetic modeling, we showed that the preferred use of glucose as a carbon source for purine ring synthesis in NSCLC tissues involves cytoplasmic activation/compartmentation of the glucose-to-serine pathway and enhanced reversed one-carbon fluxes that attenuate exogenous serine incorporation into purines. Our findings also indicate that the substrate for de novo nucleotide synthesis differs profoundly between cancer cell lines and fresh human lung cancer tissues; the latter preferred glucose to exogenous serine or glycine but not the former. This distinction in substrate utilization in purine synthesis in human cancer tissues should be considered when targeting one-carbon metabolism for cancer therapy.
| S-EPMC6737211 | biostudies-literature
Project description:Despite purines making up one of the largest classes of metabolites in a cell, little is known about the regulatory mechanisms that facilitate efficient purine production. Under conditions resulting in high purine demand, enzymes within the de novo purine biosynthetic pathway cluster into multienzyme assemblies called purinosomes. Purinosome formation has been linked to molecular chaperones HSP70 and HSP90; however, the involvement of these molecular chaperones in purinosome formation remains largely unknown. Here, we present a new-found biochemical mechanism for the regulation of de novo purine biosynthetic enzymes mediated through HSP90. HSP90-client protein interaction assays were employed to identify two enzymes within the de novo purine biosynthetic pathway, PPAT and FGAMS, as client proteins of HSP90. Inhibition of HSP90 by STA9090 abrogated these interactions and resulted in a decrease in the level of available soluble client protein while having no significant effect on their interactions with HSP70. These findings provide a mechanism to explain the dependence of purinosome assembly on HSP90 activity. The combined efforts of molecular chaperones in the maturation of PPAT and FGAMS result in purinosome formation and are likely essential for enhancing the rate of purine production to meet intracellular purine demand.
| S-EPMC6192518 | biostudies-literature
Project description:Purine nucleotides are indispensable compounds for many organisms and participate in basic vital activities such as heredity, development, and growth. Blocking of purine nucleotide biosynthesis may inhibit proliferation and development and is commonly used in cancer therapy. However, the function of the purine nucleotide biosynthesis pathway in the pathogenic fungus Magnaporthe oryzae is not clear. In this study, we focused on the de novo purine biosynthesis (DNPB) pathway and characterized MoAde8, a phosphoribosylglycinamide formyltransferase, catalyzing the third step of the DNPB pathway in M. oryzae. MoAde8 was knocked out, and the mutant (∆Moade8) exhibited purine auxotroph, defects in aerial hyphal growth, conidiation, and pathogenicity, and was more sensitive to hyperosmotic stress and oxidative stress. Moreover, ∆Moade8 caused decreased activity of MoTor kinase due to blocked purine nucleotide synthesis. The autophagy level was also impaired in ∆Moade8. Additionally, MoAde5, 7, 6, and 12, which are involved in de novo purine nucleotide biosynthesis, were also analyzed, and the mutants showed defects similar to the defects of ∆Moade8. In summary, de novo purine nucleotide biosynthesis is essential for conidiation, development, and pathogenicity in M. oryzae.
| S-EPMC9502316 | biostudies-literature
Project description:Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients.
| S-EPMC8105035 | biostudies-literature