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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Published on: October 21, 2012
Selection of apoptosis-deficient adenovirus E4orf4 mutants in Saccharomyces cerevisiae
R Afifi1, R Sharf, R Shtrichman
1Gonda Center of Molecular Microbiology, Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel.
Abstract:
Adenovirus E4orf4 protein has been shown to induce p53-independent, protein phosphatase 2A (PP2A)-dependent apoptosis in transformed cells. Furthermore, E4orf4 also induces toxicity in Saccharomyces cerevisiae in a PP2A-dependent manner (D. Kornitzer and T. Kleinberger, submitted for publication). In this work, we utilized yeast cells to select for nonapoptotic E4orf4 mutants which, in turn, were shown to possess a diminished ability to bind PP2A. The success of this selection system will provide additional apoptosis-relevant mutants for E4orf4 research and strongly supports the relevance of E4orf4-induced toxicity in S. cerevisiae to E4orf4-induced apoptosis in mammalian cells.
Insights
Adenovirus E4orf4 protein triggers cell death (apoptosis) in cancer cells and yeast. Researchers used yeast to identify E4orf4 mutants that do not cause apoptosis, revealing a reduced ability to bind protein phosphatase 2A (PP2A).
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Adenovirus E4orf4 protein induces apoptosis in transformed cells, independent of p53.
- This E4orf4-induced apoptosis is dependent on protein phosphatase 2A (PP2A).
- E4orf4 also causes toxicity in Saccharomyces cerevisiae via a PP2A-dependent mechanism.
Purpose of the Study:
- To utilize yeast as a model system to select for nonapoptotic E4orf4 mutants.
- To investigate the role of PP2A binding in E4orf4-mediated toxicity and apoptosis.
- To identify novel E4orf4 mutants for further research into apoptosis.
Main Methods:
- A selection system in yeast was employed to isolate E4orf4 mutants.
- Mutants were screened for their ability to induce apoptosis in yeast.
- The binding affinity of selected mutants to PP2A was assessed.
Main Results:
- Nonapoptotic E4orf4 mutants were successfully selected using the yeast system.
- These nonapoptotic mutants exhibited a diminished ability to bind PP2A.
- The findings support the relevance of E4orf4 toxicity in yeast to apoptosis in mammalian cells.
Conclusions:
- The yeast selection system is effective for identifying apoptosis-relevant E4orf4 mutants.
- Reduced PP2A binding correlates with a loss of E4orf4-induced toxicity and apoptosis.
- This study validates the use of yeast as a model for studying adenovirus E4orf4 function in mammalian cells.
