Abstract
The core domain of the tumor suppressor p53 has low thermodynamic stability, and many oncogenic mutations cause it to denature rapidly at body temperature. We made a series of core domain mutants that are significantly less or more stable than wild type to investigate effects of stability on the transcriptional activity and levels of native full-length p53 in H1299 mammalian cells. The levels of transcriptionally inactive native protein with inactivating mutations in the N-terminal transactivation domain correlated strongly with stability. The levels of transcriptionally active proteins, however, depended on both their stability and the transcriptional activity that leads to the feedback loop of proteolytic degradation via transcription of E3 ligases. A very highly stabilized quadruple mutant and an even more stable hexamutant were more active than wild-type p53 in terms of Bax transcription and apoptotic activity, and reached higher levels than wild type in cells. The increased activity did not result from increased overall stability but was due to a single known suppressor mutation, N239Y. It is possible that the low intrinsic stability of p53 is a means of keeping its level low in the cell by spontaneous denaturation, by a route additional to that of proteolytic degradation via E3 ligase pathways. Denatured p53 does accumulate in cells, and there are pathways for the proteolysis of denatured proteins.
Highlights
P53 contains intrinsically disordered N- and C-terminal domains, while its central core and its tetramerization domains are folded [10]
In 50% of human cancers, p53 is inactivated by oncogenic mutations [12], which are mostly found in the DNA binding core domain of p53 [13]
The level of expression of the mutant p53 core domains in Escherichia coli, in the absence of Hdm2 and other E3 ligases feedback loops that are found in mammalian cells, depend on their thermodynamic stability [26]
Summary
P53 contains intrinsically disordered N- and C-terminal domains, while its central core and its tetramerization domains are folded [10]. The stabilized quadruple mutant M133L/V203A/N239Y/ N268D core domain (QM3 or T-p53C, residues 94 –312) was designed semirationally, and found to be more stable than the wild-type p53 core domain by 2.5 kcal/mol and fully functional in vitro [18]. The level of expression of the mutant p53 core domains in Escherichia coli, in the absence of Hdm and other E3 ligases feedback loops that are found in mammalian cells, depend on their thermodynamic stability [26]. We examined the effects of thermodynamic stability of the core domain of full-length p53, by making use of the stabilized quadruple (QM) and HM, and expressing them in mammalian cells with active feedback loops involving Hdm. We examined the levels of native p53 protein and the effects of stability on transcription and apoptosis
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