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Study of purinosome assembly in cell-based model systems with de novo purine synthesis and salvage pathway deficiencies
V. Baresova, V. Skopova, O. Souckova, M. Krijt, S. Kmoch, M. Zikanova,
Jazyk angličtina Země Spojené státy americké
Typ dokumentu časopisecké články, práce podpořená grantem
Grantová podpora
NV15-28979A
MZ0
CEP - Centrální evidence projektů
Digitální knihovna NLK
Plný text - Článek
Zdroj
NLK
Directory of Open Access Journals
od 2006
Free Medical Journals
od 2006
Public Library of Science (PLoS)
od 2006
PubMed Central
od 2006
Europe PubMed Central
od 2006
ProQuest Central
od 2006-12-01
Open Access Digital Library
od 2006-01-01
Open Access Digital Library
od 2006-01-01
Open Access Digital Library
od 2006-10-01
Medline Complete (EBSCOhost)
od 2008-01-01
Nursing & Allied Health Database (ProQuest)
od 2006-12-01
Health & Medicine (ProQuest)
od 2006-12-01
Public Health Database (ProQuest)
od 2006-12-01
ROAD: Directory of Open Access Scholarly Resources
od 2006
- MeSH
- biologické modely * MeSH
- HeLa buňky MeSH
- lidé MeSH
- multienzymové komplexy genetika metabolismus MeSH
- puriny biosyntéza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
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- $a 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.
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