Corroboration of the functional role of the additional zinc binding site in the G245C mutant form of the p53 protein

Our earlier suggestion that the G245C mutation in p53 generates an additional zinc binding site, overlapping with the normal zinc binding site, has been supported by molecular modeling. The energy of interaction between a zinc ion and the new site in the G245 mutant is comparable to that for the normal site in wild-type p53. The presence of the additional site in the mutant can distort its conformation when it interacts with DNA. Effects of other mutations on the energy of zinc binding to the normal site have been calculated.     

Corroboration of the functional role of the additional zinc binding site in the G245C mutant form of the p53 protein

Our earlier suggestion that the G245C mutation in p53 generates an additional zinc binding site, overlapping with the normal zinc binding site, has been supported by molecular modeling. The energy of interaction between a zinc ion and the new site in the G245 mutant is comparable to that for the normal site in wild-type p53. The presence of the additional site in the mutant can distort its conformation when it interacts with DNA. Effects of other mutations on the energy of zinc binding to the normal site have been calculated.

     

Analysis of a protein-binding domain of p53.

The tumor suppressor protein p53 was first isolated as a simian virus 40 large T antigen-associated protein and subsequently was found to function in cell proliferation control. Tumor-derived mutations in p53 occur predominantly in four evolutionarily conserved regions spanning approximately 50% of the polypeptide. Previously, three of these regions were identified as essential for T-antigen binding. We have examined the interaction between p53 and T antigen by using Escherichia coli-expressed human p53. By a combination of deletion analysis and antibody inhibition studies, a region of p53 that is both necessary and sufficient for binding to T antigen has been localized. This function is contained within residues 94 to 293, which include the four conserved regions affected by mutation in tumors. Residues 94 to 293 of p53 were expressed in both wild-type and mutant forms. T-antigen binding was unaffected by tumor-derived mutations which have been associated with the wild-type conformation of p53 but was greatly reduced by mutations which were previously shown to alter p53 conformation. Our results show that, like T-antigen binding to the Rb tumor suppressor protein, T antigen appears to interact with the domain of p53 that is commonly mutated in human tumors.     

Discrimination of single amino acid mutations of the p53 protein by means of deterministic singularities of recurrence quantification analysis

p53 is mutated in roughly 50% of all human tumors, predominantly in the DNA-binding domain codons. Structural, biochemical, and functional studies have reported that the different p53 mutants possess a broad range of behaviors that include the elimination of the tumor-suppression function of wild-type protein, the acquisition of dominant-negative function over the wild-type form, and the establishment of gain-of-function activities. The contribution of each of these types of mutations to tumor progression, grade of malignancy, and response to anticancer treatments has been so far analyzed only for a few "hot-spots." In an attempt to identify new approaches to systematically characterize the complete spectrum of p53 mutations, we applied recurrence quantification analysis (RQA), a non-linear signal analysis technique, to p53 primary structure. Moving from the study of the p53 hydrophobicity pattern, which revealed important similarities with the singular deterministic structuring of prions, we could statistically discriminate, on a pure amino acid sequence basis, between experimentally characterized DNA-contact defective and conformational p53 mutants with a very high percentage of success. This result indicates that RQA is a mathematical tool particularly advantageous for the development of a database of p53 mutations that integrates epidemiological data with structural and functional categorizations.     

An intron binding protein is required for transformation ability of p53.

Regulatory elements in intron sequences have been identified for several eukaryotic genes. The fourth intron of p53 is known to increase expression of p53 in a position dependent manner. We asked whether p53 intron 4 sequences interacted with DNA binding proteins to exact their effect. Three overlapping DNA fragments spanning the 5' end of p53 intron 4 were determined to specifically interact with protein in nuclear extracts from several cell lines by band shift analysis. Methylation interference experiments were used to identify purine residues involved in this protein-DNA interaction. Two G nucleotides were identified at intron 4 positions 33 and 44 and these were replaced by T and C, respectively. These two single base pair substitutions in the intron resulted in 1) lack of protein binding and 2) decreased expression of p53 as measured by a transformation assay. Thus the binding of protein to p53 intron 4 was shown to have functional significance. These experiments demonstrated a specific protein binding region in the 5' end of intron 4 critical for p53 expression and distinct from those elements already known to be involved in splicing.     

Conformational effects of selected cancer-related amino acid substitutions in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Cancer-related mutations in the human p53 protein tend to cluster in four of the five highly conserved domains of the protein, and, in particular, in the central region of domain IV from residues 241 to 253. Using conformational energy analysis based on ECEPP (Empirical Conformational Energies for Polypeptides Program), we have determined the preferred three dimensional structures for this tridecapeptide sequence for the human wild-type p53 protein and four cancer-related mutant p53 proteins (Ala 245, Ile 246, Trp 248, Ser 249). The results show that the mutant peptides adopt conformations that are distinctly different from that of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in murine wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of human mutant p53 proteins may be mediated by distinct local conformational changes in the protein.     

Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53-binding protein 2 (53BP2)

The tumor suppressor p53 is frequently mutated in human cancers. Upon activation it can induce cell cycle arrest or apoptosis. ASPP2 can specifically stimulate the apoptotic function of p53 but not cell cycle arrest, but the mechanism of enhancing the activation of pro-apoptotic genes over cell cycle arrest genes remains unknown. In this study, we analyzed the binding of 53BP2 (p53-binding protein 2, the C-terminal domain of ASPP2) to p53 core domain and various mutants using biophysical techniques. We found that several p53 core domain mutations (R181E, G245S, R249S, R273H) have different effects on the binding of DNA response elements and 53BP2. Further, we investigated the existence of a ternary complex consisting of 53BP2, p53, and DNA response elements to gain insight into the specific pro-apoptotic activation of p53. We found that binding of 53BP2 and DNA to p53 is mutually exclusive in the case of GADD45, p21, Bax, and PIG3. Both pro-apoptotic and non-apoptotic response elements were competed off p53 by 53BP2 with no indication of a ternary complex.     

Mutant <Emphasis Type="Italic">TP53</Emphasis> G245C and R273H promote cellular malignancy in esophageal squamous cell carcinoma

Background

TP53 gene mutations occur in more than 50% of human cancers and the vast majority of these mutations in human cancers are missense mutations, which broadly occur in DNA binding domain (DBD) (Amino acids 102–292) and mainly reside in six “hotspot” residues. TP53 G245C and R273H point mutations are two of the most frequent mutations in tumors and have been verified in several different cancers. In the previous study of the whole genome sequencing (WGS), we found some mutations of TP53 DBD in esophageal squamous cell carcinoma (ESCC) clinical samples. We focused on two high-frequent mutations TP53 p.G245C and TP53 p.R273H and investigated their oncogenic roles in ESCC cell lines, p53-defective cell lines H1299 and HCT116 p53?/?.

Results

MTS and colony formation assays showed that mutant TP53 G245C and R273H increased cell vitality and proliferation. Flow cytometry results revealed inhibition of ultraviolet radiation (UV)- and ionizing radiation (IR)- induced apoptosis and disruption of TP53-mediated cell cycle arrest after UV, IR and Nocodazole treatment. Transwell assays indicated that mutant TP53 G245C and R273H enhanced cell migration and invasion abilities. Moreover, western blot revealed that they were able to suppress the expression of TP53 downstream genes in the process of apoptosis and cell cycle arrest induced by UV, which suggests that these two mutations can influence apoptosis and growth arrest might be due, at least in part, to down-regulate the expression of P21, GADD45α and PARP.

Conclusions

These results indicate that mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy, which further uncover TP53 function in carcinogenesis and might be useful in clinical diagnosis and therapy of TP53 mutant cancers.

     

Recognition of DNA by p53 core domain and location of intermolecular contacts of cooperative binding

We present an analysis by NMR of a 58 kDa complex of the core domain of the tumour suppressor p53 with DNA that complements and extends the crystal structure analysis. Binding of specific DNA caused significant chemical shifts of residues on the DNA-binding interface that translated into the beta-sheet of the protein. Binding of non-specific DNA caused weak but qualitatively the same shifts, corresponding to weaker binding interactions. The observed chemical shift differences correlate with frequency of cancer-inducing mutations, suggesting that the affected residues contribute to the stability of p53 core domain-DNA complex. We also identified two affected regions on the surface of the protein: helix 1 (residues V173-C182) plus G244 and residues L114-T118, which may represent a dimerisation interface.     

Common conformational effects of p53 mutations

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Most of these mutations occur in highly conserved regions in the DNA-binding core domain of the p53 protein, suggesting that the amino acid residues in these regions are critical for maintaining normal p53 structure and function. We previously used molecular dynamics calculations to demonstrate that several amino acid substitutions in these regions that are induced by environmental carcinogens and found in human tumors produce certain common conformational changes in the mutant proteins that differ substantially from the wild-type structure. In order to determine whether these conformational changes are consistent for other p53 mutants, we have now used molecular dynamics to determine the structure of the DNA-binding core domain of seven other environmentally induced, cancer-related p53 mutants, namely His 175, Asp 245, Asn 245, Trp 248, Met 249, Ser 278, and Lys 286. The results indicate that all of these mutants differ substantially from the wild-type structure in certain discrete regions and that some of these conformational changes are similar for these mutants as well as those determined previously. The changes are also consistent with experimental evidence for alterations in structure in p53 mutants determined by epitope detectability using monoclonal antibodies directed against these regions of predicted conformational change.     

Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation.

In an effort to correlate the biological activity of the p53 protein with its conformation, we analysed 14 p53 mutants representative of the most frequently observed protein alterations in human cancers, at codons 175, 248 and 273 (22% of all mutations thus far reported), all three of which contained a CpG dinucleotide. Strikingly, most of the mutants at codons 248 and 273 did not display any change in their conformation, as probed by monoclonal antibodies PAb240 and PAb1620 or by binding to hsp70 protein. For all 14 mutants tested, we found a strict correlation between the transactivation properties of p53, tested either on RGC sequences or using the WAF-1 promoter, and inhibition of cell proliferation. All these mutants showed nuclear localization. Several mutants, present at a low incidence in human tumours, displayed wild-type activity in all our assays, suggesting that the presence of a mutation is not strictly correlated with p53 protein inactivation in tumours. Further analysis of nine thus far undescribed p53 mutants at codon 175 revealed a wild-type or mutant behaviour. All these results suggest that the occurrence of a mutation is dependent on two criteria: (i) the mutability of a given codon, such as those containing a CpG dinucleotide; (ii) the resulting amino acids, eventually leading to synthesis of a p53 conferring a growth advantage on the cell.     

Critical amino acids of p21 protein are located within beta-turns: further evaluation

It has been shown that malignant activation of ras proto-oncogenes was mediated by point mutations which resulted in the single amino acid conversions at positions 12, 13 or 61 of the ras gene products (p21 proteins). By analyzing randomly mutated ras genes, it has been demonstrated that amino acid substitutions at residues 12, 13, 59 and 63 activated p21. Furthermore, it has been shown that residues 16, 116 and 119 in p21 played critical roles in the guanine nucleotide binding and, consequently, the ability of the protein to induce changes characteristic of cellular transformation. By using the protein conformational prediction method of Chou and Fasman, the present work predicts that these critical amino acids, except glutamic acid at position 63, are located within beta-turns. The major "hot spots" for ras activation are codons 12 and 61. The author has predicted in an earlier paper that the single amino acid conversions at positions 12 and 61 would occur at beta-turn conformation consisting of residues 10-13 and 58-61, respectively. In the present study, probabilities of beta-turn occurrence at residues 10-13 or 58-61 of the p21 proteins encoded by various ras genes are compared. The probability for the normal p21 containing glycine as residue 12 is greatest, and the cancer-associated variants show less probabilities. The single amino acid substitutions at position 61 do not cause so decreased probabilities of beta-turn potential at residues 58-61, except the replacement by histidine. Histidine at position 61 is not predicted as occurring within a beta-turn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maximum likelihood analysis of mammalian p53 indicates the presence of positively selected sites and higher tumorigenic mutations in purifying sites

The tumor suppressor gene TP53 (p53) maintains genome stability. Mutation or loss of p53 is found in most cancers. Analysis of evolutionary constrains and p53 mutations reveal important sites for concomitant functional studies. In this study, phylogenetic analyses of the coding sequences of p53 from 26 mammals were carried out by applying a maximum likelihood method. The results display two branches under adaptive evolution in mammals. Moreover, each codon of p53 was analyzed by the PAML method for presence of positively selected sites. PAML identified several statistically significant amino acids that undergo positive selection. The data indicates that amino acids responsible for the core functions of p53 are highly conserved, while positively selected sites are predominantly located in the N- and C-terminus of p53. Further analysis of evolutionary pressure and mutations showed the occurrence of more frequent tumorigenic mutations in purifying sites of p53.