Cancer-associated p53 Tetramerization Domain Mutants

Abstract

The tumor suppressor p53, a 393-amino acid transcription factor, induces cell cycle arrest and apoptosis in response to genotoxic stress. Its inactivation via the mutation of its gene is a key step in tumor progression, and tetramer formation is critical for p53 post-translational modification and its ability to activate or repress the transcription of target genes vital in inhibiting tumor growth. About 50% of human tumors have TP53 gene mutations; most are missense ones that presumably lower the tumor suppressor activity of p53. In this study, we explored the effects of known tumor-derived missense mutations on the stability and oligomeric structure of p53; our comprehensive, quantitative analyses encompassed the tetramerization domain peptides representing 49 such substitutions in humans. Their effects on tetrameric structure were broad, and the stability of the mutant peptides varied widely (⌬T m ‫؍‬ 4.8 ϳ ؊46.8°C). Because formation of a tetrameric structure is critical for protein-protein interactions, DNA binding, and the post-translational modification of p53, a small destabilization of the tetrameric structure could result in dysfunction of tumor suppressor activity. We suggest that the threshold for loss of tumor suppressor activity in terms of the disruption of the tetrameric structure of p53 could be extremely low. However, other properties of the tetramerization domain, such as electrostatic surface potential and its ability to bind partner proteins, also may be important.

Quantitative Correlation between the Protein Expression Level inEscherichia Coliand Thermodynamic Stability of Protein In Vitro

“…The relative expression levels of the p53TD mutants compared with WT were analyzed using the ¦G u values at 37°C, which were calculated from the in vitro thermal denaturation analysis data. 16 As shown in Figure 2, the expression level of the p53TD mutants strongly correlated with the thermodynamic stability of their tetramer. In the case of p53TD mutants with minimal to moderate destabilization, the correlation between expression levels and their ¦G u values was approximately proportional.…”

“…The relative expression level to WT is plotted as a function of the ¦G u value at 37°C, which was calculated using data of a previous study. 16 Each point is shown by Entry No. as listed in Table 1.…”

“…The structural features of p53TD have been well characterized in many studies, including comprehensive Ala-scanning and intensive mutational analyses. 15,16 Thermodynamic analysis has revealed that missense mutations of p53TD in tumors destabilized the tetramer formation. 16 The destabilization effects of the mutations ranged from minimal to severe.…”

“…15,16 Thermodynamic analysis has revealed that missense mutations of p53TD in tumors destabilized the tetramer formation. 16 The destabilization effects of the mutations ranged from minimal to severe. p53TD missense mutations have the least effects on mRNA stability, because of a point mutation and a simple protein folding pathway other than thermodynamic stability, which are important factors for protein expression in bacterial cells.…”

“…However, with the bacterial expression system there are still difficulties with expressing and obtaining recombinant proteins, because of the poor growth of the host cell, inclusion bodies formation, protein inactivity, and sometimes not obtaining any protein at all. 16 Several studies have reported that the protein expression level of recombinant proteins in E. coli depends on protein synthesis and degradation, which are regulated by various factors such as mRNA stability, differences in codon usage between prokaryotes and eukaryotes, and the protein folding state. 712 In general, it is thought that the expression of proteins with low structural stability is often difficult.…”