CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation

A missense mutation in PFAS (phosphoribosylformylglycinamidine synthase) is likely causal for embryonic lethality associated with the MH1 haplotype in Montbéliarde dairy cattle

“…Implantation of homozygous mutants and control embryos, close monitoring of gestations, and gene expression analyses would be required to test these hypotheses and further understand molecular mechanisms causing the death of homozygous mutants. Finally, mutations affecting genes belonging to the purine de novo and salvage synthesis pathways are investigated as potential candidates for severe metabolic disorders in humans, which are supposed to cause prenatal or early death in their most severe and hardly detectable forms (Baresova et al, 2016). Interestingly, we found in the human Exome Aggregation Consortium database the same p.R1205C substitution in the PFAS protein (GRCh37/hg19; Chr17: 8172081C>T; Lek et al, 2016), which segregates at a very low frequency in European and Asian populations (4 alleles over 117,110 observed; no homozygote).…”

A New View into the Regulation of Purine Metabolism: The Purinosome

“…Probing with the remaining four pathway enzymes also showed co-clustering with FGAMS [6]. Immunofluorescence has demonstrated purinosome formation on the endogenous level and provided evidence that the observed compartmentalization is not a consequence of overexpression due to transient transfection [6, 34-36]. Additionally, particle characterization in transient transfected models demonstrated that these enzyme clusters are distinct in size and cell density from processing bodies (P-bodies), stress granules, and aggresomes (Box 2) [37].…”

“…Recently, mutations in ADSL and ATIC observed in skin fibroblasts from patients with AICAR-ribosiduria and ADSL deficiency showed decrease purinosome formation suggesting that activity of these periphery purinosome enzymes impact complex stability (Table 1) [35]. Likewise, HeLa cell lines deficient in specific pathway enzymes resulted either in a complete loss of purinosomes (GART, ADSL, ATIC) or a significant reduction (FGAMS, PAICS) compared to normal HeLa cells [34]. Measurement of the diffusion coefficients of these enzymes in Hs578T breast carcinoma cells using fluorescence recovery after photobleaching substantiated the spatial model of core and peripheral enzymes that assemble stepwise (Figure 2B) [43].…”

“…Recent studies have shown that deficiency in enzymatic activity of metabolic enzymes impact metabolon formation [34, 35]. These data argue that enzymatic activity of the individual components are important for complex stability; however, enzymatic activity is not likely the only criterion for complex formation.…”

Spatial Organization of Metabolic Enzyme Complexes in Cells

“…91,92 Furthermore, knockout of any purine biosynthetic enzyme resulted in either the reduction or abolishment of purinosome association in human cells. 93 More recently, the functional activity of mechanistic target of rapamycin (mTOR) was linked to the spatial association of purinosomes with the mitochondria as well as purine biosynthesis. 94,95 Collectively, the formation of purinosomes, which indicates the upregulation of de novo purine biosynthesis, has significantly advanced our understanding of the regulatory mechanisms of de novo purine biosynthesis in human cells.…”