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Disruption of functions of wild-type p53 by hetero-oligomerization
D Deb1, A S Chakraborti, A Lányi
1Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA.
Abstract:
We report that a p53 segment (p53 del 1-293) containing the oligomerization domain interferes with the functions of wild-type p53. Wild-type p53 inhibits transcription mediated by human cytomegalovirus (CMV) immediate-early promoter significantly; however, co-expression of p53 del 1-293 drastically reduces this repression. We show that wild-type p53 forms hetero-oligomers with p53 del 1-293 suggesting that the hetero-oligomers are defective in repressing the CMV promoter. A synthetic promoter with p53-binding sites is transactivated significantly by wild-type p53. However, co-expression of p53 del 1-293 drastically reduces this activation. At a high concentration, a deletion mutant of wild-type p53 (del 393-327) defective in oligomerization transactivates efficiently a promoter with synthetic p53-binding sites. This transactivation remains unaffected by co-expression of p53 del 1-293. p53 del 393-327 also fails to hetero-oligomerize with p53 del 1-293 indicating that hetero-oligomerization is necessary for disruption of wild-type p53-mediated transactivation. Immunostaining experiments show that hetero-oligomerization does not lead to changes in localization of nuclear p53 demonstrating that delocalization of p53 is not the reason for inactivation. We also show that co-expression of p53 del 1-293 significantly reduces the G1/S arrest by wild-type p53 suggesting that a proper oligomeric form is necessary for wild-type p53-mediated cell cycle arrest. Thus, our work shows that hetero-oligomerization disrupts wild-type p53's biological functions and suggests a mechanism by which
Insights
A p53 segment disrupts wild-type p53 functions by forming non-functional hetero-oligomers. This interference impairs p53
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The tumor suppressor protein p53 plays a critical role in cellular responses to stress.
- p53 functions as a transcription factor that regulates genes involved in cell cycle arrest, apoptosis, and DNA repair.
- The oligomerization domain of p53 is essential for its transcriptional activity and formation of functional complexes.
Purpose of the Study:
- To investigate the mechanism by which a specific p53 deletion mutant (p53 del 1-293), containing the oligomerization domain, interferes with wild-type p53 function.
- To determine if hetero-oligomerization between wild-type p53 and the deletion mutant is responsible for the observed functional disruption.
- To elucidate the role of p53 oligomerization in transcriptional regulation and cell cycle control.
Main Methods:
- Co-expression of wild-type p53 with p53 deletion mutants in cellular systems.
- Reporter gene assays using the human cytomegalovirus (CMV) immediate-early promoter and synthetic p53-binding sites.
- Analysis of p53 hetero-oligomerization using co-immunoprecipitation or similar techniques.
- Immunostaining to assess the subcellular localization of p53.
- Cell cycle analysis to evaluate the impact on G1/S arrest.
Main Results:
- Co-expression of p53 del 1-293 significantly reduced wild-type p53-mediated repression of the CMV promoter and transactivation of synthetic p53-binding site promoters.
- Wild-type p53 formed hetero-oligomers with p53 del 1-293, and these complexes were defective in transcriptional repression and activation.
- A deletion mutant defective in oligomerization (p53 del 393-327) efficiently transactivated promoters and was unaffected by co-expression of p53 del 1-293, indicating hetero-oligomerization is key to disruption.
- Hetero-oligomerization did not alter the nuclear localization of p53.
- Co-expression of p53 del 1-293 significantly reduced wild-type p53-mediated G1/S cell cycle arrest.
Conclusions:
- Hetero-oligomerization between wild-type p53 and the p53 del 1-293 mutant disrupts the biological functions of wild-type p53.
- The formation of non-functional hetero-oligomers is a likely mechanism for dominant-negative effects of certain p53 mutants.
- Proper oligomeric structure of p53 is essential for its transcriptional activity and cell cycle arrest functions.