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Characterization of the p53 mutants ability to inhibit p73 beta transactivation using a yeast-based functional assay
Paola Monti1, Paola Campomenosi, Yari Ciribilli
1Mutagenesis Laboratory, National Cancer Research Institute (IST), Genova, Italy.
Abstract:
p53 is the most frequently altered tumor suppressor gene in a wide spectrum of human tumors. The large majority of p53 mutations observed in tumors are missense mutations. The p73 gene, encoding a protein with significant sequence similarity to p53, expresses multiple transcription-competent spliced variants, or transcription-incompetent forms (i.e. DeltaNp73). It was clearly shown that p73 transactivation from a p53-responsive promoter is inhibited by some tumor-derived p53 mutants in eucaryotic cells. In this study, we adapted a yeast-based p53 functional assay for the analysis of the influences of different p53 mutants on the activity of one of the p73 isoforms, namely p73beta. We determined the ability of a panel of 61 p53 mutants to inhibit p73beta activity following the net transcription of the ADE2 color (red/white) reporter gene driven by a p53-responsive promoter. By analysing a large number of mutants, we could conclude that interference: (a) is a quite frequent phenomenon (more than 70% of p53 mutants analysed are interfering); (b) is not confined to p53 mutations located in particular topological regions of the DNA binding domain; (c) does not appear to be dependent on the kind of side chains introduced at a specific position; (d) appears to significantly correlate with evolutionary conservation of the mutated p53 codon, frequency of occurrence of the mutation in tumors. The influence of a common R/P polymorphism at codon 72 on the ability of p53 mutants to interfere with p73beta was also studied. Two sets of polymorphic variants (R and P) for 14 mutants were constructed and analysed. In all cases, the R/P 72 polymorphism was phenotypically irrelevant. In conclusion, our results suggest that the interpretation of the biological effects of p53 mutants should take into consideration the possibility that p53 mutants show loss or gain of function also through the interference with p53 family members.
Insights
Most p53 mutations interfere with p73beta activity, suggesting a gain of function for these tumor suppressor gene variants. This interference is frequent and linked to evolutionary conservation and tumor occurrence, impacting p53 family interactions.
Area of Science:
- Molecular Biology
- Cancer Genetics
- Tumor Suppressor Genes
Background:
- TP53 is the most frequently mutated tumor suppressor gene in human cancers, with missense mutations being predominant.
- The p53 family member, p73, shares sequence similarity with p53 and has multiple spliced variants, including transcription-competent and incompetent forms.
- Previous studies indicated that certain tumor-derived p53 mutants can inhibit p73 transactivation.
Purpose of the Study:
- To adapt a yeast-based functional assay for analyzing the influence of p53 mutants on p73beta activity.
- To determine the interference capability of 61 different p53 mutants with p73beta.
- To investigate the correlation between p53 mutant interference and mutation characteristics, including location, conservation, and tumor frequency.
Main Methods:
- A yeast functional assay was employed to assess the impact of 61 p53 mutants on p73beta activity.
- Reporter gene expression (ADE2 color) driven by a p53-responsive promoter was used to quantify p73beta transactivation inhibition.
- The influence of the p53 codon 72 R/P polymorphism on mutant interference was analyzed using constructed polymorphic variants.
Main Results:
- Over 70% of the analyzed p53 mutants demonstrated interference with p73beta activity, indicating a frequent phenomenon.
- Interference was not restricted to specific topological regions of the p53 DNA-binding domain or dependent on the nature of introduced side chains.
- Mutant interference significantly correlated with the evolutionary conservation of the mutated p53 codon and its frequency in tumors. The codon 72 R/P polymorphism showed no phenotypic relevance.
Conclusions:
- Many p53 mutants exhibit a gain of function through interference with p53 family members like p73beta.
- The biological effects of p53 mutants should be interpreted considering their potential to interfere with p53 family interactions.
- This study highlights a novel mechanism by which p53 mutations contribute to tumorigenesis beyond simple loss of function.