Modified HeLa Cells and C.elegans Multicellular Model as Unique Systems for the Study of Purinosome Formation

Abstract

Purines are essential molecules for synthesis of nucleic acids, universal carriers of chemical energy and parts of signaling molecules in all living organisms. Their cellular level is maintained by the de novo purine synthesis (DNPS), effective recycling by salvage pathway and eventual degradation. DNPS includes ten reactions catalyzed by six enzymes, four of them are multifunctional in high eukaryotes. Some DNPS intermediates are unstable and/or toxic, therefore the proximity of enzymes in multienzyme complex called purinosome would be essential to ensure the vital metabolic function. The first direct evidence of its formation was demonstrated by detecting spatial overlap of signals of transiently expressed fluorescently labeled DNPS proteins in HeLa cells grown in the purine depleted medium. However, this model and its eventual utility for further research was questioned and attributed to aggregation of overexpressed proteins and stress granules resulting from the exposure of the cells to the dialyzed, and therefore nutrient reduced growth media. Another possibility for detection of purinosome complexes is immunofluorescent labeling of endogenous proteins involved in the DNPS pathway, so the artificial protein overexpression is avoided. The disadvantage of this method is the inability to study purinosome formation in vivo.In this work we detected the purinosome by combining two previously used methods in prepared purinosome non‐forming cells deficient for particular DNPS steps (CR‐DNPS cells) and in cells deficient in salvage pathway which build purinosome regardless to the purine levels (CR‐HGPRT cells). Moreover, we have prepared the model organism C. elegans with fluorescently labeled DNPS enzymes for in vivo study of the purinosome formation.We transiently transfected the CR‐DNPS and CR‐HGPRT cells with relevant vectors coding BFP labeled wild‐type proteins and observed normalization (e.g. restoration or disruption) of purinosome formation in the cells.To control if the cells undergoing transient transfection and nutrition starvation formed purinosomes or stress bodies, we transfected one type of CR‐DNPS cells with vector coding enzyme with mutation resulting in zero enzyme activity. We did not find any purinosomes regardless of the quantity of purines in the growth media.We fused DNPS proteins in C. elegans with fluorescent protein GFP or mCherry and observed the intracellular compartmentalization of purinosome by fluorescencent confocal microscopy.In conclusion, both methods, transient transfection and immunofluorescence, are useful for the detection of purinosomes in HeLa cells. Moreover, prepared cell‐based models and multicellular model organism C. elegans with translational reporters represent a unique system for purinosome assembly studies. From these models, we expect a greater understanding of the complexity of purine metabolism.Support or Funding InformationThis work was supported by grants AZV 15‐28979A (Ministry of Health, CR); PRIMUS/17/MED/6, GAUK 1102217 and PROGRES Q26/LF1 (Charles University, CR); and LQ1604 NPU II (Ministry of Education, Youth and Sports, CR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.