Transcriptome and metabolome analysis of crGART, a novel cell model of de novo purine synthesis deficiency: Alterations in CD36 expression and activity

Abstract

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.

The Purinosome: A Case Study for a Mammalian Metabolon

“…Under physiological conditions, 5-PRA exhibits a relatively short half-life (5 s at 37°C) in the absence of the subsequent pathway enzyme GART, thus disallowing accumulation of 5-PRA. This finding was recently confirmed in HeLa cells in which expression of GART, the 5-PRA processing enzyme, was knocked out, and 5-PRA accumulation was not observed (39). Although these studies provide a rationale for the sequestration of 5-PRA and its efficient conversion into the downstream stable metabolite glycinamide ribonucleotide (GAR) by GART, in vitro protein-protein interaction studies did not support this proposition (38).…”