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

• DOI: 10.1096/fasebj.2018.32.1_supplement.527.16
• Publication Date: 2021-06-21T16:23:16Z
• Authors: Vaclava Skopova; Veronika Baresova; Olga Souckova; Matyas Krijt; Marie Zikanova
• Keywords: Nucleotide salvage; Purine metabolism; HeLa

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