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Regulation of mutant p53 temperature-sensitive DNA binding
P Friedlander1, Y Legros, T Soussi
1Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Abstract:
We have examined in detail the DNA binding properties of several immunopurified tumor-derived mutant p53 proteins (Val-143 --> Ala, Arg-175 --> His, Arg-248 --> Trp, Arg-249 --> Ser, and Arg-273 --> His). While all mutants were defective for binding to DNA at 37 ;C, each bound specifically to several cognate p53 binding sites at sub-physiological temperatures (25-33 ;C), and several mutants activated transcription from a p53-responsive promoter at 26 degrees C in transfected H1299 cells. Heating mutant p53 proteins at 37 degrees C irreversibly destroyed their ability to subsequently bind at 25 degrees C. However, several different monoclonal antibodies that each share the ability to recognize an epitope encompassing amino acids 46-55 markedly stabilized binding by mutant p53 proteins at 37 degrees C. Both intact antibody and FAb fragments allowed mutant p53 to bind to DNA. By contrast, antibodies that recognize epitopes located elsewhere within p53 stabilized mutant p53 binding significantly less effectively. Our data show that the major hot-spot p53 mutants have the intrinsic ability to bind to DNA and that a unique region within the N terminus of p53 may be critical for rescuing them from loss of binding at physiological temperatures. This suggests the possibility of developing small molecules that can stabilize mutant p53 proteins under physiological conditions.
Insights
Mutant tumor suppressor p53 proteins can bind DNA at lower temperatures, but lose this ability when heated. Specific antibodies targeting an N-terminal epitope can restore DNA binding at physiological temperatures.
Area of Science:
- Molecular Biology
- Cancer Research
- Protein Biochemistry
Background:
- Mutations in the tumor suppressor gene p53 are common in human cancers.
- Understanding the DNA binding properties of mutant p53 is crucial for cancer therapy.
Purpose of the Study:
- To investigate the DNA binding capabilities of various tumor-derived mutant p53 proteins.
- To explore methods for restoring the DNA binding function of mutant p53.
Main Methods:
- Immunopurification of tumor-derived mutant p53 proteins.
- Assessment of DNA binding at different temperatures (25-37°C).
- Analysis of transcriptional activation in transfected cells.
- Evaluation of antibody stabilization of mutant p53 DNA binding.
Main Results:
- Mutant p53 proteins exhibited defective DNA binding at 37°C but bound specifically at lower temperatures (25-33°C).
- Heating mutant p53 irreversibly abolished DNA binding at lower temperatures.
- Monoclonal antibodies recognizing an N-terminal epitope (amino acids 46-55) significantly stabilized mutant p53 binding at 37°C.
- Antibody fragments also restored DNA binding, unlike antibodies targeting other epitopes.
Conclusions:
- Major hot-spot p53 mutants retain the intrinsic ability to bind DNA.
- An N-terminal region of p53 is critical for maintaining DNA binding at physiological temperatures.
- Developing small molecules to stabilize mutant p53 at physiological temperatures is a potential therapeutic strategy.