In humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We selected the Cluster of Differentiation (CD36) gene for initial analysis based on its elevated expression in crGART versus HeLa as well as its high basal expression, high log2 value, and minimal P-value.

• MeSH
• Phosphoribosylglycinamide Formyltransferase metabolism   genetics   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Metabolome * MeSH
• Purines * metabolism   biosynthesis   MeSH
• Transcriptome * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phosphoribosylglycinamide Formyltransferase MeSH
• purine MeSH Browser
• Purines * MeSH

Adenylosuccinate lyase (ADSL) catalyzes two steps in de novo purine synthesis (DNPS). Mutations in ADSL can result in inborn errors of metabolism characterized by developmental delay and disorder phenotypes, with no effective treatment options. Recently, SAICAR, a metabolic substrate of ADSL, has been found to have alternative roles in the cell, complicating the role of ADSL. crADSL, a CRISPR KO of ADSL in HeLa cells, was constructed to investigate DNPS and ADSL in a human cell line. Here we employ this cell line in an RNA-seq analysis to initially investigate the effect of DNPS and ADSL deficiency on the transcriptome as a first step in establishing a cellular model of ADSL deficiency. We report transcriptome changes in genes relevant to development, vascular development, muscle, and cancer biology, which provide interesting avenues for future research.

• Keywords
• Adenylosuccinate lyase, Purine synthesis, RNA-seq, Transcriptome, adenosine monophosphate, (AMP), adenylosuccinate lyase, (ADSL), aminoimidazole carboxamide ribonucleotide, (AICAR), de novo purine synthesis, (DNPS), differentially expressed gene, (DEG), false discovery rate, (FDR), fetal calf macroserum, (FCM), fragments per kilobase of exon per million reads mapped, (FPKM), gene ontology, (GO), guanosine monophosphate, (GMP), minus adenine crADSL to minus adenine WT comparison, (MM), phosphoribosyl pyrophosphate, (PRPP), phosphoribosylaminoimidazolesuccinocarboxamide, (SAICAR), plus adenine crADSL to plus adenine WT comparison, (PP), succinyladenosine monophosphate, (SAMP),
• Publication type
• Journal Article MeSH

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.

• MeSH
• Chromatography, Liquid MeSH
• CRISPR-Cas Systems MeSH
• DNA biosynthesis   chemistry   MeSH
• Gene Editing MeSH
• HeLa Cells MeSH
• Humans MeSH
• Purines biosynthesis   chemistry   MeSH
• RNA biosynthesis   chemistry   MeSH
• Tandem Mass Spectrometry * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• purine MeSH Browser
• Purines MeSH
• RNA MeSH