Quantitative analysis of purine nucleotides indicates that purinosomes increase de novo purine biosynthesis

Abstract

Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome.

Keywords: Mass Spectrometry (MS); Metabolic Flux; Metabolomics; Nucleoside/Nucleotide Biosynthesis; Protein Complex; Purine; Purinosome.

FIGURE 1.

a, immunofluorescence imaging of endogenous PPAT and FGAMS colocalization in fixed HeLa cells. HeLa cells cultured in purine-depleted growth media were fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100 prior to being stained with Atto488-labeled PPAT and Cy3-labeled FGAMS antibodies. The degree of colocalization of PPAT and FGAMS was determined by Pearson's linear correlation coefficient: 0.93. Scale bar: 10 μm. b, the ratios of cellular concentrations of ATP/ADP and AMP/GMP remain the same between the two culture conditions. c, the IMP level of HeLa cells treated with DMAT is about 1.5 times higher than normal HeLa cells. *, p < 0.01 (Student's t test) n = 10. Data are presented as the mean ± S.D.

FIGURE 2.

Relative abundance of intercellular purines in ¹H NMR metabolite scanning (AMP, 5′-IMP, guanosine, adenosine, hypoxanthine, and inosine) in HeLa cells cultured in purine-depleted medium (shown in blue) and purine-rich medium (shown in red). Data are presented as the mean ± S.D. *, p < 0.05, **, p < 0.01, ***, p < 0.001 (2-tailed Student's t test), n = 10.

FIGURE 3.

Simplified IMP biosynthesis and catabolic pathways in humans. IMP can be synthesized from PRPP by the de novo purine biosynthesis pathway or made from hypoxanthine by the salvage pathway. IMP can be further transformed into AMP and GMP or recycled back by AMP deaminase and GMP reductase. ADSL, adenylosuccinate lyase; GMPS, GMP synthase.

FIGURE 4.

Incorporation of [¹⁵N]glycine into purines by the de novo biosynthesis pathway. a, the incorporation of [¹⁵N]glycine into IMP. The incorporation was plotted using percentage of ¹⁵N incorporated purines as a function of time of incubation. HeLa cells cultured in P− media (in blue) showed a faster incorporation rate than cells in P+ media (in red) within 1.5 h. The initial incorporation rate for IMP, AMP, and GMP was calculated according to the slope of the linear relationship of the incorporation plot within 1 h. The initial rate of cells in purine-depleted condition was increased by ∼47% for IMP biosynthesis. b, the incorporation of [¹⁵N]glycine into AMP. c, the incorporation of [¹⁵N]glycine into GMP. Data are presented as the mean ± S.D.

FIGURE 5.

Colocalization of transiently transfected ADSS-GFP or IMPDH-GFP with FGAMS-OFP in HeLa cells grown in purine-depleted media and formation of IMPDH filaments (rods) in HeLa cells. a–f, ADSS-GFP (b) and IMPDH-GFP (e) were co-transfected with FGAMS-OFP (a and d); c and f are merged images. The degree of colocalization was determined by the Pearson's linear correlation coefficient: 0.88 (ADSS and FGAMS) and 0.82 (IMPDH and FGAMS). g, IMPDH-GFP transiently transfected HeLa cells showing the filament or rod structures, as indicated by white arrows. h, the filament macrostructure formed in the right cell (white arrows) is different from the purinosome spots (red arrows) in the left cell. All images were taken on the Nikon TE-2000E inverted fluorescence microscope. Scale bar, 10 μm.

FIGURE 6.

Western blot analysis of enzymes in the purine de novo biosynthesis pathway in HeLa cells. Quantification of protein expression levels from three independent blots was determined by calculating the optical density of the target blot. There was no difference in levels for five out of six enzymes between cells cultured in P+ and P− media. ATIC in cells under P− condition has higher expression according to the Student's t test between P+ and P− conditions (p < 0.05). GAPDH or β-actin was used as the loading control. PAICS, carboxyaminoimidazole ribonucleotide synthase-succinyl aminoimidazole carboxamide ribonucleotide synthetase; TriGART, trifunctional GARS-GAR transformylase-aminoimidazole ribonucleotide synthetase; ASL, adenylosuccinate lyase.