Tracing the evolution of the p53 tetramerization domain.
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ABSTRACT:
SUBMITTER: Joerger AC
PROVIDER: S-EPMC4155161 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compensating effects of up to 9.5 kcal/mol of tetramer. The triple mutant L332V/F341L/L344I, whose hydrophobic core represents that of the chicken p53 domain, was nearly as stable as the human domain but had impaired hetero-oligomerization with it. Thus, engineering of a functional p53 variant with a reduced capacity to hetero-oligomerize with wild-type human p53 can be achieved without any impairment in the stability and subunit affinity of the engineered homo-oligomer.
| S-EPMC22339 | biostudies-literature
Project description:Functional in a tetrameric state, the protein product of the p53 tumor suppressor gene confers its tumor-suppressive activity by transactivating genes which promote cell-cycle arrest, senescence, or programmed cell death. How p53 distinguishes between these divergent outcomes is still a matter of considerable interest. Here we discuss the impact of 2 mutations in the tetramerization domain that confer unique properties onto p53. By changing lysines 351 and 357 to arginine, thereby blocking all post-translational modifications of these residues, DNA binding and transcriptional regulation by p53 remain virtually unchanged. On the other hand, by changing these lysines to glutamine (2KQ-p53), thereby neutralizing their positive charge and potentially mimicking acetylation, p53 is impaired in the induction of cell cycle arrest and yet can still effectively induce cell death. Surprisingly, when 2KQ-p53 is expressed at high levels in H1299 cells, it can bind to and transactivate numerous p53 target genes including p21, but not others such as miR-34a and cyclin G1 to the same extent as wild-type p53. Our findings show that strong induction of p21 is not sufficient to block H1299 cells in G1, and imply that modification of one or both of the lysines within the tetramerization domain may serve as a mechanism to shunt p53 from inducing cell cycle arrest.
| S-EPMC4934057 | biostudies-literature
Project description:Triple negative breast cancer (TNBC) has no targeted detection or treatment method. Mutant p53 (mtp53) is overexpressed in >80% of TNBCs, and the stability of mtp53 compared to the instability of wild-type p53 (wtp53) in normal cells makes mtp53 a promising TNBC target for diagnostic and theranostic imaging. We generated Cy5p53Tet, a novel nucleus-penetrating mtp53-oligomerization-domain peptide (mtp53ODP) to the tetramerization domain (TD) of mtp53. This mtp53ODP contains the p53 TD sequence conjugated to a Cy5 fluorophore for near-infrared fluorescence imaging (NIRF). In vitro co-immunoprecipitation and glutaraldehyde cross-linking showed a direct interaction between mtp53 and Cy5p53Tet. Confocal microscopy and flow cytometry demonstrated higher uptake of Cy5p53Tet in the nuclei of TNBC MDA-MB-468 cells with mtp53 R273H than in ER-positive MCF7 cells with wtp53. Furthermore, depletion of mtp53 R273H caused a decrease in the uptake of Cy5p53Tet in nuclei. In vivo analysis of the peptide in mice bearing MDA-MB-468 xenografts showed that Cy5p53Tet could be detected in tumor tissue 12 min after injection. In these in vivo experiments, significantly higher uptake of Cy5p53Tet was observed in mtp53-expressing MDA-MB-468 xenografts compared with the wtp53-expressing MCF7 tumors. Cy5p53Tet has clinical potential as an intraoperative imaging agent for fluorescence-guided surgery, and the mtp53ODP scaffold shows promise for modification in the future to enable the delivery of a wide variety of payloads including radionuclides and toxins to mtp53-expressing TNBC tumors.
| S-EPMC8068092 | biostudies-literature
Project description:The tumor suppressor function of the wild-type p53 protein is transdominantly inhibited by tumor-derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild-type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild-type p53 subunits into inactive wild-type/mutant hetero-tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor-derived mutant p53, would escape transdominant inhibition. Aided by the known three-dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full-length p53 protein bearing this novel domain formed homo-tetramers and had tumor suppressor function, but did not hetero-oligomerize with tumor-derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain.
| S-EPMC2144399 | biostudies-other
Project description:A highly-parallel yeast functional assay, capable of screening approximately 100-1,000 mutants in parallel and designed to screen the activity of transcription activator proteins, was utilized to functionally characterize tetramerization domain mutants of the human p53 transcription factor and tumor suppressor protein. A library containing each of the 19 possible single amino acid substitutions (57 mutants) at three positions in the tetramerization domain of the human p53 protein, was functionally screened in Saccharomyces cerevisiae. Amino acids Leu330 and Ile332, whose side chains form a portion of a hydrophobic pocket that stabilizes the active p53 tetramer, were found to tolerate most hydrophobic amino acid substitutions while hydrophilic substitutions resulted in the inactivation of the protein. Amino acid Gln331 tolerated essentially all mutations. Importantly, highly parallel mutagenesis and cloning techniques were utilized which, in conjunction with recently reported highly parallel DNA sequencing methods, would be capable of increasing throughput an additional 2-3 orders of magnitude.
| S-EPMC2269032 | biostudies-other
Project description: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 ? -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.
| S-EPMC3012982 | biostudies-literature
Project description:We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency-lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.
| S-EPMC5565410 | biostudies-literature
Project description:The p53 tumor suppressor is a tetrameric transcriptional enhancer, and its activity is compromised by mutations that cause amino acid substitutions in its tetramerization domain. Here we analyze the biochemical and biophysical properties of peptides corresponding to amino acids 319-358 of wild-type human p53, which includes the tetramerization domain, and that of a cancer-derived mutant with valine substituted for glycine 334. Unlike the wild-type peptide, the G334V peptide forms amyloid fibrils by a two-step process under physiological conditions of temperature and pH. Nevertheless, the G334V peptide is capable of forming heterooligomers with a wild-type peptide. Computational modeling of the G334V peptide structure suggests that substitution of valine for glycine 334 causes a local distortion that contributes to a beta-dominated structural transition leading to amyloid formation. Since the distortion is mostly on the surface, the mutant peptide is still able to form a pseudonative tetramer complex at higher concentrations and/or lower temperatures. Our study suggests a new potential mechanism by which mutations that compromise tetramer formation inactivate p53 as a tumor suppressor.
| S-EPMC2536691 | biostudies-literature
Project description:The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA-binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation present in the population in south Brazil. In this work, we combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. In addition to the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt-bridge interaction that stabilizes the p53 tetramerization domain were studied (i.e., R337D, D352R, and the double-mutation R337D plus D352R). The simulations revealed a destabilizing effect of the pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay, performed in yeast by tuning the expression of wild-type and mutant p53 proteins, revealed that p53 tetramerization mutations could decrease the transactivation potential and alter transactivation specificity, in particular by better tolerating negative features in weak DNA-binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53's conformational stability and function.
| S-EPMC9316806 | biostudies-literature
Project description:Protein p53 is a transcription factor crucial for cell cycle and genome integrity. It is able to induce both cell arrest when DNA is damaged and the expression of DNA repair machinery. When the damage is irreversible, it triggers apoptosis. Indeed, the protein, which is a homotetramer, is mutated in most human cancers. For instance, the inherited mutation p53-R337H results in destabilization of the tetramer and, consequently, leads to an organism prone to tumor setup. We describe herein a rational designed molecule capable of holding together the four monomers of the mutated p53-R337H protein, recovering the tetramer integrity as in the wild-type structure. Two ligand molecules, based on a conical calix[4]arene with four cationic guanidiniomethyl groups at the wider edge (upper rim) and hydrophobic loops at the narrower edge (lower rim), fit nicely and cooperatively into the hydrophobic clefts between two of the monomers at each side of the protein and keep the tetrameric structure, like molecular templates, by both ion-pair and hydrophobic interactions. We found a good agreement between the structure of the complex and the nature of the interactions involved by a combination of theory (molecular dynamics) and experiments (circular dichroism, differential scanning calorimetry and (1)H saturation transfer difference NMR).
| S-EPMC2575436 | biostudies-literature