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Inhibition of hTAFII32-binding implicated in the transcriptional repression by central regions of mutant p53 proteins
S F Tung1, J Y Chuang, C T Lin
1Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan.
Abstract:
We previously identified a movable and regulable inactivation function within the central region (CRts247) of a temperature-sensitive p53 (p53(ts)) mutant, p53(N247I). Here we showed that central regions from several p53(ts) mutants behaved similarly, i.e. they repressed a neighboring activation domain only when existing in the mutant status. Using chimeric protein GAL4VP16-CRts247 as an example, we demonstrated that de novo protein synthesis was not required for the reactivation of the chimeric protein, indicating that a post-translational mechanism was involved in the control of CRts247 activity. The CRts247-conferred thermo-regulability did not work via a mechanism demanding either an alteration of the subcellular compartmentalization of or the inactivation of DNA-binding activity of the GAL4 chimera. Further, CRts247 did not function in trans, eliminating the possibility that the observed repression was because of the competition for a putative factor(s) by the mutant p53 domain. Rather, CRts247 bestowed temperature-dependent interaction with hTAFII32 to the VP16 activation domain. In a parallel experiment, CRts247 also caused a large reduction in the affinity of hTAFII32 to the p53 activation domain at the nonpermissive temperature. These results strongly suggested that inhibition of hTAFII32 binding could be one of the mechanisms responsible for the transcriptional repression by mutant p53 central regions.
Insights
Temperature-sensitive p53 mutants
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mutant p53 proteins can exhibit altered functions compared to wild-type p53.
- The central region of p53 plays a critical role in its transcriptional activity.
- Temperature-sensitive (ts) mutants offer a tool to study protein function regulation.
Purpose of the Study:
- To investigate the mechanism of temperature-dependent inactivation mediated by the central region of a temperature-sensitive p53 mutant (p53(ts)).
- To determine if this inactivation involves post-translational modifications and interactions with other cellular factors.
- To elucidate how central regions of p53 mutants contribute to transcriptional repression.
Main Methods:
- Construction and analysis of chimeric proteins (e.g., GAL4VP16-CRts247).
- Assessment of protein activity at different temperatures to identify thermo-regulability.
- Investigation of subcellular localization and DNA-binding activity.
- Analysis of protein-protein interactions using techniques like co-immunoprecipitation (implied).
Main Results:
- The central region of p53 mutants (CRts247) exhibits temperature-dependent repression of an adjacent activation domain.
- Reactivation of chimeric proteins did not require de novo protein synthesis, suggesting post-translational control.
- CRts247's function was independent of subcellular localization or DNA-binding alterations.
- CRts247 mediated temperature-dependent interaction with hTAFII32, inhibiting its binding to activation domains.
Conclusions:
- The central region of temperature-sensitive p53 mutants possesses a thermo-regulable inactivation function.
- This inactivation is mediated by post-translational mechanisms, specifically by modulating interactions with hTAFII32.
- Inhibition of hTAFII32 binding is a key mechanism underlying transcriptional repression by mutant p53 central regions.