Abstract
Conformational stability of the p53 protein is an absolute necessity for its physiological function as a tumor suppressor. Recent in vitro studies have shown that wild-type p53 is a highly temperature-sensitive protein at the structural and functional levels. Upon heat treatment at 37 degrees C, p53 loses its wild-type (PAb1620(+)) conformation and its ability to bind DNA, but can be stabilized by different classes of ligands. To further investigate the thermal instability of p53, we isolated p53 mutants resistant to heat denaturation. For this purpose, we applied a recently developed random mutagenesis technique called DNA shuffling and screened for p53 variants that could retain reactivity to the native conformation-specific anti-p53 antibody PAb1620 upon thermal treatment. After three rounds of mutagenesis and screening, mutants were isolated with the desired phenotype. The isolated mutants were translated in vitro in either Escherichia coli or rabbit reticulocyte lysate and characterized biochemically. Mutational analysis identified 20 amino acid residues in the core domain of p53 (amino acids 101-120) responsible for the thermostable phenotype. Furthermore, the thermostable mutants could partially protect the PAb1620(+) conformation of tumor-derived p53 mutants from thermal unfolding, providing a novel approach for restoration of wild-type structure and possibly function to a subset of p53 mutants in tumor cells.
Highlights
Activation of the p53 tumor suppressor protein appears to be an integrating mechanism in response to cellular stresses such as DNA damage and oxidative stress [1,2,3]
A more quantitative approach regarding the stability of p53 has been performed using differential scanning calorimetry, where the core domains of wild-type p53 and several tumor-derived p53 mutants were subjected to urea-mediated denaturation [30]
The results of this study demonstrated that the core domain of p53 is of moderate thermodynamic stability, with all the tested mutant core domains being less stable than the wild type
Summary
Activation of the p53 tumor suppressor protein appears to be an integrating mechanism in response to cellular stresses such as DNA damage and oxidative stress [1,2,3]. Point mutations in the p53 gene have been identified in 50% of human tumors, indicating that p53 inactivation is an important step in tumor progression [17,18,19] The majority of these mutations (Ͼ90%) cluster in the central core domain of the protein and are responsible for the loss of the biological activity of p53 [20, 21]. Similar studies have been performed on tumor-derived p53 mutants, which appear to be even less thermostable than wild-type p53, but again, N-terminal anti-p53 antibodies can partially protect these mutants from temperature-induced unfolding [29]. A subset of the p53 mutations found in tumors destabilize the folded state of the protein, affecting the normal biological function of p53
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
