BACKGROUND: The enzymes involved in de novo purine synthesis (DNPS), one of the basic processes in eukaryotic cells, transiently and reversibly form a dynamic multienzyme complex called the purinosome in the cytoplasm. The purinosome has been observed in a broad spectrum of cells, but some studies claim that it is an artefact of the constructs used for visualization or stress granules resulting from the exposure of cells to nutrient-reduced growth media. Both may be true depending on the method of observation. To clarify this point, we combined two previously used methods, transfection and immunofluorescence, to detect purinosomes in purinosome-free cells deficient in particular DNPS steps (CR-DNPS cells) and in cells deficient in the salvage pathway, which resulted in construction of the purinosome regardless of purine level (CR-HGPRT cells). METHODS AND FINDINGS: To restore or disrupt purinosome formation, we transiently transfected CR-DNPS and CR-HGPRT cells with vectors encoding BFP-labelled wild-type (wt) proteins and observed the normalization of purinosome formation. The cells also ceased to accumulate the substrate(s) of the defective enzyme. The CR-DNPS cell line transfected with a DNA plasmid encoding an enzyme with zero activity served as a negative control for purinosome formation. No purinosome formation was observed in these cells regardless of the purine level in the growth medium. CONCLUSION: In conclusion, both methods are useful for the detection of purinosomes in HeLa cells. Moreover, the cell-based models prepared represent a unique system for the study of purinosome assembly with deficiencies in DNPS or in the salvage pathway as well as for the study of purinosome formation under the action of DNPS inhibitors. This approach is a promising step toward the treatment of purine disorders and can also provide targets for anticancer therapy.

Research Unit for Rare Diseases Department of Paediatrics and Adolescent Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague Czech Republic

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Jurecka A, Zikanova M, Kmoch S, Tylki-Szymanska A. Adenylosuccinate lyase deficiency. J Inherit Metab Dis. 2015; 38(2):231–42. 10.1007/s10545-014-9755-y PubMed DOI PMC

Pedley AM, Benkovic SJ. A New View into the Regulation of Purine Metabolism: The Purinosome. Trends Biochem Sci. 2017;42(2):141–54. 10.1016/j.tibs.2016.09.009 PubMed DOI PMC

An S, Kumar R, Sheets ED, Benkovic SJ. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science. 2008;320(5872):103–6. 10.1126/science.1152241 PubMed DOI

Zhao A, Tsechansky M, Ellington AD, Marcotte EM. Revisiting and revising the purinosome. Mol Biosyst. 2014;10(3):369–74. PubMed PMC

An S, Deng Y, Tomsho JW, Kyoung M, Benkovic SJ. Microtubule-assisted mechanism for functional metabolic macromolecular complex formation. Proc Natl Acad Sci U S A. 2010;107(29):12872–6. 10.1073/pnas.1008451107 PubMed DOI PMC

French JB, Jones SA, Deng H, Pedley AM, Kim D, Chan CY, et al. Spatial colocalization and functional link of purinosomes with mitochondria. Science. 2016;351(6274):733–7. 10.1126/science.aac6054 PubMed DOI PMC

Chan CY, Zhao H, Pugh RJ, Pedley AM, French J, Jones SA, et al. Purinosome formation as a function of the cell cycle. Proc Natl Acad Sci U S A. 2015;112(5):1368–73. 10.1073/pnas.1423009112 PubMed DOI PMC

Baresova V, Skopova V, Sikora J, Patterson D, Sovova J, Zikanova M, et al. Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency. Hum Mol Genet. 2012;21(7):1534–43. 10.1093/hmg/ddr591 PubMed DOI

Baresova V, Krijt M, Skopova V, Souckova O, Kmoch S, Zikanova M. CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation. Mol Genet Metab. 2016;119(3):270–7. 10.1016/j.ymgme.2016.08.004 PubMed DOI

Zikanova M, Skopova V, Hnizda A, Krijt J, Kmoch S. Biochemical and structural analysis of 14 mutant adsl enzyme complexes and correlation to phenotypic heterogeneity of adenylosuccinate lyase deficiency. Hum Mutat. 2010;31(4):445–55. 10.1002/humu.21212 PubMed DOI

Kmoch S, Hartmannova H, Stiburkova B, Krijt J, Zikanova M, Sebesta I. Human adenylosuccinate lyase (ADSL), cloning and characterization of full-length cDNA and its isoform, gene structure and molecular basis for ADSL deficiency in six patients. Hum Mol Genet. 2000;9(10):1501–13. PubMed

Landmann L. Deconvolution improves colocalization analysis of multiple fluorochromes in 3D confocal data sets more than filtering techniques. J Microsc. 2002;208(Pt 2):134–47. PubMed

Manders EMM, Verbeek FJ, Aten JA. Measurement of colocalization of objects in dual-color confocal images. J Microsc-Oxford. [Article]. 1993;169:375–82. PubMed

Fu R, Sutcliffe D, Zhao H, Huang X, Schretlen DJ, Benkovic S, et al. Clinical severity in Lesch-Nyhan disease: the role of residual enzyme and compensatory pathways. Mol Genet Metab. 2015;114(1):55–61. 10.1016/j.ymgme.2014.11.001 PubMed DOI PMC

Parker WB. Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev. 2009;109(7):2880–93. 10.1021/cr900028p PubMed DOI PMC

Mass spectrometric analysis of purine de novo biosynthesis intermediates