Oligomerization Domain Of P53 Research Articles

High-throughput evaluation of genetic variants with prime editing sensor libraries.

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

DARPins detect the formation of hetero-tetramers of p63 and p73 in epithelial tissues and in squamous cell carcinoma

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p632/p732 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p632/p732 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.

Abstract 5644: Activation of p53 in prolonged prometaphase

Abstract The tumor suppressor p53 plays a critical role in protecting cells from aberrant divisions in the face of DNA damage, and while its role in DNA damage checkpoints have been extensively studied, its role in the mitotic checkpoint is less well characterized. The mitotic checkpoint prevents completion of mitosis until chromosomes have made bipolar kinetochore attachments to microtubules. Previous studies have shown that cells that experience a prolonged prometaphase(>1.5 h) following treatment with microtubule destabilizing drugs will arrest in G1 following eventual exit from mitosis. Given its role in DNA damage checkpoints, p53 appears to be a likely candidate for involvement in this arrest. The goal of the current study is to investigate the role of p53 in this G1 arrest following prolonged prometaphase. Three cells lines were utilized:MCF10A, MCF10A/αp53 which overexpressed shRNA to p53, and MCF10A/OD which overexpresses the p53 oligomerization domain. Cells were treated with 9 μM RO-3306 for 18 h to synchronize cells at the G2/M transition. The RO-3306 was then removed and cells were treated with a microtubule destabilizing drug, nocodazole, at a concentration of 0.08 μM for 1.5 or 2.5 h to cause prometaphase arrest. The cells were then washed to remove nocodazole, and the cells were harvested at various time points and analyzed via flow cytometry and Western blot. Flow cytometry analysis in in all three cell lines showed an accumulation of cells in G1 at 12 hours in for both treatment times (1.5 and 2.5 h), with subsequent increases in sub-G1 populations by 24 hours. Western blot analysis revealed activation of p53 by phosphorylation of ser20 and activation of p21 in the MCF10A and MCF10A/OD, but not in the MCF10A/αp53 for both treatment times. Taken together, these results suggest that activation of p53 alone is not sufficient to elicit the G1 arrest, as the MCF10A/αp53 cells arrested in G1 despite the absence of p53 protein. Citation Format: Megan Anderson, Aime A. Levesque. Activation of p53 in prolonged prometaphase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5644.

Mouse Homolog of the Human TP53 R337H Mutation Reveals Its Role in Tumorigenesis.

Inheritance of germline mutations in the tumor suppressor gene TP53 causes Li-Fraumeni syndrome (LFS), a cancer predisposition disorder. The arginine to histidine substitution at amino acid position 337 of p53 (R337H) is a founder mutation highly prevalent in southern and southeastern Brazil and is considered an LFS mutation. Although this mutation is of significant clinical interest, its role in tumorigenesis using animal models has not been described. Here, we generate a knockin mouse model containing the homologous R337H mutation (mouse R334H). De novo tumorigenesis was not significantly increased in either heterozygous (p53334R/H ) or homozygous (p53334H/H ) p53 R334H knockin mice compared with wild-type mice. However, susceptibility to diethylnitrosamine (DEN)-induced liver carcinogenesis was increased in a mutant allele dose-dependent manner. In parallel, p53334H/H mice exposed to DEN exhibited increased DNA damage but decreased cell-cycle regulation in the liver. Oligomerization of p53, which is required for transactivation of target genes, was reduced in R334H liver, consistent with its decreased nuclear activity compared with wild-type. By modeling a TP53 mutation in mice that has relatively weak cancer penetrance, this study provides in vivo evidence that the human R337H mutation can compromise p53 activity and promote tumorigenesis.Significance: A germline mutation in the oligomerization domain of p53 decreases its transactivation potential and renders mice susceptible to carcinogen-induced liver tumorigenesis. Cancer Res; 78(18); 5375-83. ©2018 AACR.

Abstract P2-07-04: HER2 overexpressing breast cancers are more frequent in carriers of TP53 DNA-binding domain mutations than in carriers of oligomerization-site mutations

Abstract Germline TP53 mutations are associated with Li-Fraumeni Syndrome (LFS), an autosomal dominant disorder characterized by predisposition to multiple early-onset cancers including breast cancer (BC), the most prevalent tumor among women. The majority of germline TP53 mutations are missense substitutions clustered within the DNA-binding domain of the gene that disrupt the structure and function of the protein. Recently, the germline mutation p.R337H (c.1010G>A), lying in the oligomerization domain of p53, was reported at a high frequency in Southern and Southeastern Brazil. This mutation appears to result in a more subtle defect in the protein, which becomes functionally deficient only under particular conditions and specific tissues. Recent reports suggest that BC are HER2-postive in a high proportion (63-83%) of TP53 mutation carriers. Histopathologic features of breast cancers among carriers of oligomerization domain mutations have not been reported. Our objective was to analize HER2 status in breast cancers from carriers of germline TP53 mutations, both in the DNA-binding and oligomerization domains. We retrospectively reviewed the immunohistochemistry data of breast cancers from 64 carriers of TP53 p.R337H and 6 carriers of nonfunctional germline TP53 mutations (p.G245S, p.R273H, p.G244D or c.672+1G>T). All patients were recruited from the cancer genetics clinic at Hospital de Clínicas de Porto Alegre, Brazil. Germline TP53 mutations were identified by Sanger sequencing of the entire coding sequence and flanking intronic regions. Immunohistochemistry (IHC) was performed using standard procedures. HER2 expression was assessed using a 0 to 3+ scale according the ASCO-CAP Guidelines. All of the six patients with nonfunctional TP53 mutations showed strong (3+) HER2 staining by IHC in their breast tumors. In the group of p.R337H carriers, 23.4% showed significant HER2 overexpression (3+), 21.9% presented ambiguous HER2 results (2+), and 54.7% showed weak or no HER2 expression (1+ or 0). Thus, HER2 overexpressing breast cancers were more frequent in carriers of TP53 nonfunctional (DNA binding domain) mutations (p≤0.001) than in those carrying the oligomerization domain mutation. Preliminary results from this study suggest that germline mutations in different domains of the TP53 gene may predispose to breast cancer through different mechanisms. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-04.

Abstract 3771: Establishment of primary cultures of cancer stem cells from patients with Li-Fraumeni Syndrome, carrier of TP53 p.R337H Brazilian founder mutation.

Abstract Li-Fraumeni syndrome (LFS), an inherited cancer predisposition syndrome, is associated with germline mutations in TP53. It is characterized by high risk of multiple, early cancers. In Brazil, a variant form of LFS is exceedingly frequent due to a widespread founder TP53 mutation, p.R337H, detected in about 0.3% of the general population in Southern Brazil. This mutation occurs in p53 oligomerization domain and its effect on p53 oligomerization is supposed to be dependent upon pH conditions. Recent studies indicate that p53 plays a critical role in regulating differentiation and asymmetric divisions of stem cells. The main objective of this study is to isolate and to characterize Cancer Stem Cells (CSC) in patients with germline mutations in TP53 gene with Li-Fraumeni syndrome and Li-Fraumeni-like Syndrome. We have isolated and characterized CSC from tumors of p.R337H mutation carriers. After informed consent, surgical resection fragments were dissociated and brought in culture. Adherent cells and spheroids were derived from different tumor types. Spheroids and stromal cells derived from a breast cancer (BC) were further analyzed by immunofluorescence and flow cytometry to demonstrate positive immunolabeling for CD44+, CD24−, Oct4, Ki67 and Sox2 antibodies. Time-lapse videomicroscopy showed rapid growth, frequent asymmetric division and absence of senescent phenotypes for least 17 passages. Moreover, we showed by Colony Forming Units assay (CFU) that stromal cells are very clonogenic, once 103 cells initially plated were able to form 442 new colonies after 15 days in culture. It was also verified the differentiation potential of stromal cells for the following lineages: adipogenic, osteogenic and muscle-like cells. The adherent stromal cells as well as oncoesferas are able to self-renew and to proliferate in vitro for long periods without losing its main features. These properties are similar to stem cell possessing the p53 inactive. However, we observed that treatment of adherent cells with doxorubicin (DNA damaging agent) causes accumulation of p53 and causing induction of cell cycle arrest with low doses of this drug, and cell death / apoptosis in higher doses. Our findings using CSC-p.R337H can provide a very useful model for better understanding the role of specific mutation, as well as the TP53 tumor biology. Citation Format: Lisley I. Mambelli, Irina Kerkis, Pierre Hainaut, Maria Isabel W. Achatz. Establishment of primary cultures of cancer stem cells from patients with Li-Fraumeni Syndrome, carrier of TP53 p.R337H Brazilian founder mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3771. doi:10.1158/1538-7445.AM2013-3771

A novel approach to cancer treatment using structural hybrids of the p53 gene family

The p53 tumor suppressor belongs to a gene family that includes two other structurally and functionally related members: p73 and p63. The regulation of p53 activity differs significantly from that of p73 and p63. To enhance the tumor suppressive activity of p53, we constructed six recombinant adenoviruses that encode hybrid proteins with three functional domains derived from either p53 or TAp63γ. The potency of these hybrid molecules in suppressing tumorigenesis was evaluated using in vitro and in vivo models. Of the hybrid molecules tested, one hybrid named p63-53O was the most potent activator of apoptosis in human cancer cells. The p63-53O hybrid is composed of the transcriptional activation domain and DNA-binding domain of TAp63γ and the oligomerization domain of p53. The p63-53O hybrid efficiently transactivated p53AIP1. Moreover, silencing of p53AIP1 partially abolished the apoptotic response to p63-53O in human cancer cells. The p53-p63 hybrid molecule is a novel potent anti-proliferative agent for the treatment of cancer.

Abstract 5349: Cancer stem cells isolation and characterization from breast tumor of a germline carrier of TP53 p.R337H (Brazilian founder) mutation

Abstract Li-Fraumeni syndrome (LFS), an inherited cancer predisposition syndrome, is associated with germ line mutations in TP53. It is characterized by high risk of multiple, early cancers. In Brazil, a variant form of LFS is exceedingly frequent due to a widespread founder TP53 mutation, p.R337H, detected in about 0.3% of the general population in Southern Brazil. This mutation occurs in p53 oligomerization domain and its effect on p53 oligomerization is supposed to be dependent upon pH conditions. We have isolated and characterized Cancer Stem Cells from tumors of p.R337H mutation carriers. After informed consent and surgical resection fragments were dissociated and brought in culture. Adherent cells and spheroids were derived from different tumor types. Spheroids derived from a breast cancer (BC) were further analyzed to demonstrate positive immunolabeling for CD44, CD24, Oct4 and Sox2 antibodies. Time-lapse videomicroscopy showed rapid growth, frequent asymmetric division and absence of senescent phenotypes for least 17 passages. At confluence, some cells spontaneously differentiated into adipogenic and muscle-like cells. Floating and adherent spheroids contained self-renewing cells capable of long-term proliferation without losing their primary characteristics. These properties are similar to those of stem cells with inactivated p53 protein. However, treatment of adherent cells with the DNA damaging agent doxorubicin elicited p53 accumulation, p21 induction, cell cycle arrest at low doses of Dox and cell death/apoptosis at higher doses. These results are consistent with the notion that p.R337H may retain a quasi-normal p53 function in response to stress while expressing properties similar to mutant p53 in long-term CSC culture. These p.R337H CSC may provide a very useful model for studying the defects underlying inherited cancer in p.R337H carriers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5349. doi:1538-7445.AM2012-5349

KR-POK Interacts with p53 and Represses Its Ability to Activate Transcription of p21WAF1/CDKN1A

Transcriptional regulation by p53 is thought to play a role in its ability to suppress tumorigenesis. However, there remain gaps in understanding about how p53 regulates transcription and how disrupting this function may promote cancer. Here we report a role in these processes for the kidney cancer-related gene KR-POK (ZBTB7C), a POZ domain and Krüppel-like zinc finger transcription factor that we found to physically interact with p53. Murine embryonic fibroblasts isolated from genetically deficient mice (Kr-pok(-/-) MEFs) exhibited a proliferative defect relative to wild-type mouse embryonic fibroblasts (MEF). The zinc finger domain of Kr-pok interacted directly with the DNA binding and oligomerization domains of p53. This interaction was essential for Kr-pok to bind the distal promoter region of the CDKN1A gene, an important p53 target gene encoding the cell-cycle regulator p21WAF1, and to inhibit p53-mediated transcriptional activation of CDKN1A. Kr-pok also interacted with the transcriptional corepressors NCoR and BCoR, acting to repress histone H3 and H4 deacetylation at the proximal promoter region of the CDKN1A gene. Importantly, Kr-pok(-/-) MEFs displayed an enhancement in CDKN1A transactivation by p53 during the DNA damage response, without any parallel changes in transcription of either the p53 or Kr-pok genes themselves. Furthermore, Kr-pok promoted cell proliferation in vitro and in vivo, and its expression was increased in more than 50% of the malignant human kidney cancer cases analyzed. Together, our findings define KR-POK as a transcriptional repressor with a pro-oncogenic role that relies upon binding to p53 and inhibition of its transactivation function.

Tracing the protectors path from the germ line to the genome

One of the basic principles that nature uses in evolution is to recycle successful concepts and create new functions by modifying existing units. This conservatism in evolution has resulted in an astonishingly high sequence identity of genes, even between evolutionarily distant species such as the nematode Caenorhabditis elegans and Homo sapiens. The recycling of successful concepts in conjunction with gene duplication events has also led to the existence of highly homologous proteins within the genome of many species. Often, these homologous proteins show similar, yet distinct functions that, in combination with their individual tissue distribution, define their specific physiological role. One prominent example is the p53 protein family, which consists of p53, p63, and p73. Recent advances in understanding the specific biological functions of these members have shed some light onto the evolution of this crucial protein family, from a germ line-specific quality-control factor to a somatic tumor suppressor. Furthermore, structures of the oligomerization domains of the mammalian paralogs, p53 and p73, and invertebrate orthologs, CEP-1 and DMP53, have delineated evolutionary changes and revealed that the oligomerization domain of p53 lacks additional stabilizing structural elements present in all other p53 family members. This suggests that p53 is the most recent evolutionary member of this protein family and predicts a mechanism for p53 activation.

Conformational stability and activity of p73 require a second helix in the tetramerization domain

p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional alpha-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family.

The tetramerization domain of p53 is required for efficient BAK oligomerization

In addition to a well-defined transcriptional activity that is necessary for efficient apoptosis induction, the p53 tumor suppressor also has a direct apoptogenic role at the mitochondria. This direct role in cell death is mediated at least in part by interaction of p53 with BCL2 family members, including the pro-apoptotic protein BAK. Whereas it is currently accepted that the mitochondrial function of p53 contributes to its tumor suppressive role, the regulation of p53 function at this organelle is poorly understood. In this manuscript we examine the role of p53 oligomerization in the regulation of its pro-apoptotic function at the mitochondria, specifically in regard to its ability to induce BAK oligomerization. We find that deletion or mutation of p53's oligomerization domain markedly impairs the ability of this protein to oligomerize BAK. Along these lines, cross-linking studies indicate that the majority of p53 localized to mitochondria is in dimeric or higher-order oligomeric form. In support of the importance of the p53-BAK interaction in the localization of p53 to mitochondria, we find that mouse embryo fibroblasts from the BAK null mouse have greatly reduced mitochondrial p53 compared to wild type fibroblasts. These data indicate that pro-apoptotic BAK, unlike other BCL2 family members, may serve as a major receptor for p53 on the mitochondria.

P53-dependent Down-regulation of Telomerase Is Mediated by p21waf1

Inactivation of p53 and activation of telomerase occur in the majority of human cancers, raising the possibility of a link between these two pathways. Overexpression of wild-type p53 down-regulates the enzymatic activity of telomerase in various cancer cell lines through transcriptional repression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In this study, we re-evaluated the role of p53 in telomerase regulation using isogenic cell lines expressing physiological levels of p53. We demonstrate that endogenous wild-type p53 was able to down-regulate telomerase activity, hTERT mRNA levels, and promoter activity; however, the ability to repress hTERT expression was found to be cell type-specific. The integrity of the DNA-binding core domain, the N-terminal transactivation domain, and the C-terminal oligomerization domains of p53 was essential for hTERT promoter repression, whereas the proline-rich domain and the extreme C terminus were not required. Southwestern and chromatin immunoprecipitation experiments demonstrated lack of p53 binding to the hTERT promoter, raising the possibility of an indirect repressive mechanism. The down-regulation of hTERT promoter activity was abolished by a dominant-negative E2F1 mutant. Mutational analysis identified a specific E2F site responsible for p53-mediated repression. Knockdown of the key p53 transcriptional target, p21, was sufficient to eliminate the p53-dependent repression of hTERT. Inactivation of the Rb family using either viral oncoproteins or RNA interference attenuated the repression. Inhibition of histone deacetylases also interfered with the repression of hTERT by p53. Therefore, our results suggest that repression of hTERT by endogenous p53 is mediated by p21 and E2F.

Dimerization of the p53 Oligomerization Domain: Identification of a Folding Nucleus by Molecular Dynamics Simulations

Dimerization of the p53 oligomerization domain involves coupled folding and binding of monomers. To examine the dimerization, we have performed molecular dynamics (MD) simulations of dimer folding from the rate-limiting transition state ensemble (TSE). Among 799 putative transition state structures that were selected from a large ensemble of high-temperature unfolding trajectories, 129 were identified as members of the TSE via calculation of a 50% transmission coefficient from at least 20 room-temperature simulations. This study is the first to examine the refolding of a protein dimer using MD simulations in explicit water, revealing a folding nucleus for dimerization. Our atomistic simulations are consistent with experiment and offer insight that was previously unobtainable.

P53 down‐regulates matrix metalloproteinase‐1 by targeting the communications between AP‐1 and the basal transcription complex

We have previously reported that human matrix metalloproteinase-1 (MMP1) is a p53 target gene subject to down-regulation (Sun et al. [1999]: J Biol Chem 274:11535-11540]. In the present study, we demonstrate that the down-regulation of the human -83MMP1 promoter fragment by p53 was abolished when the -72AP-1 site was eliminated and that a GAL4-cJun-mediated but not a GAL4-Elk1-mediated induction of pFR-luci was effectively inhibited by p53 suggesting an AP-1 dependent but AP-1 binding independent mechanism. Results from gel mobility shift assays were consistent with an AP-1 binding independent mechanism. We also demonstrate that both p300 and TATA box binding proteins cooperated with the transcription factor AP-1 to induce the promoter of MMP1; however, p53 only inhibited the p300-mediated induction of the MMP1 promoter and the inhibition was -72AP-1 dependent. Furthermore, the down-regulation of the MMP1 promoter and mRNA by p53 could be reversed by p300 and by a p53 binding p300 fragment that had no coactivator activity. Taken together, these results indicate that p53 down-regulates MMP1 mainly by disrupting the communications between the transactivator AP-1 and the basal transcriptional complex, which are partially mediated by p300. Finally, by using p53 truncated mutant constructs, we demonstrate that both the N-terminal activation domain and the C-terminal oligomerization domains of p53 were required for the down-regulation of MMP1 transcription.

Activation of the DNA-binding ability of latent p53 protein by protein kinase C is abolished by protein kinase CK2.

p53 is one of the most important regulators of cell proliferation and differentiation and of programmed cell death, triggering growth arrest and/or apoptosis in response to different cellular stress signals. The sequence-specific DNA-binding function of p53 protein can be activated by several different stimuli that modulate the C-terminal domain of this protein. The predominant mechanism of activation of p53 sequence-specific DNA binding is phosphorylation at specific sites. For example, phosphorylation of p53 by PKC (protein kinase C) occurs in undamaged cells, resulting in masking of the epitope recognized by monoclonal antibody PAb421, and presumably promotes steady-state levels of p53 activity in cycling cells. In contrast, phosphorylation by cdk2 (cyclin-dependent kinase 2)/cyclin A and by the protein kinase CK2 are both enhanced in DNA-damaged cells. We determined whether one mechanism to account for this mutually exclusive phosphorylation may be that each phosphorylation event prevents modification by the other kinase. We used non-radioactive electrophoretic mobility shift assays to show that C-terminal phosphorylation of p53 protein by cdk2/cyclin A on Ser315 or by PKC on Ser378 can efficiently stimulate p53 binding to DNA in vitro, as well as binding of the monoclonal antibody Bp53-10, which recognizes residues 371-380 in the C-terminus of p53. Phosphorylation of p53 by CK2 on Ser392 induces its DNA-binding activity to a much lower extent than phosphorylation by cdk2/cyclin A or PKC. In addition, phosphorylation by CK2 strongly inhibits PKC-induced activation of p53 DNA binding, while the activation of p53 by cdk2/cyclin A is not affected by CK2. The presence of CK2-mediated phosphorylation promotes PKC binding to its docking site within the p53 oligomerization domain, but decreases phosphorylation by PKC, suggesting that competition between CK2 and PKC does not rely on the inhibition of PKC-p53 complex formation. These results indicate the crucial role of p53 C-terminal phosphorylation in the regulation of its DNA-binding activity, but also suggest that antagonistic relationships exist between different stress signalling pathways.

A meanfield approach to the thermodynamics of a protein‐solvent system with application to the oligomerization of the tumor suppressor p53

AbstractThe thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met‐340 and Leu‐344 of human p53 were designed to disrupt the hydrophobic dimer‐dimer interface of the tetrameric oligomerization domain of p53. Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state [tetramer, dimer, or equilibrium mixture of both] of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met [tetramer] > Ser Asp, His, Gin, > Glu, Lys [dimer], whereas the experimental results showed the following order: Met [tetramer] > Ser > Gln > His, Lys > Asp, Glu [dimers]. For residue 344, the calculated trend was Leu [tetramer] > Ala > Arg, Gln, Lys [dimer], and the experimental trend was Leu [tetramer] > Ala, Arg, Gln, Lys [dimer]. The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild‐type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α‐helices at the dimer‐dimer interface. This initial application of meanfield theory toward a protein‐solvent system is encouraging for the application of the theoretical model to more complex systems.

Hetero-oligomerization does not compromise 'gain of function' of tumor-derived p53 mutants.

Tumor-derived p53 mutants activate transcription from promoters of various growth-related genes. We tested whether this transactivation function of the mutant protein is sufficient to induce tumorigenesis ('gain of function'). Tumor-derived mutant p53-281G transactivates the promoters of human epidermal growth factor receptor (EGFR) and human multiple drug resistance gene (MDR-1). To determine whether the C-terminal domain functions only as an oligomerization domain in mutant p53-mediated transactivation, we have replaced the tetramerization domain of p53 by a heterologous tetramerization domain; although this mutant protein formed tetramers in solution, it failed to transactivate significantly. Therefore, for successful mutant p53-mediated transactivation, sequences near the C-terminus of mutant p53 are required to perform functions in addition to tetramerization. We also demonstrate that co-expression of a deletion mutant of p53 (p53 del 1-293), which retains the p53 oligomerization domain, inhibits this transactivation. p53 del 1-293 co-immunoprecipitates with p53-281G suggesting that hetero-oligomers of p53-281G and p53 del 1-293 are defective in transactivation. We also show that a cell line stably transfected with p53-281G expresses higher levels of endogenous NF-kappaB and proliferating cell nuclear antigen (PCNA) compared to that transfected with vector alone. On co-expression, p53 del 1-293 lowered the levels of NF-kappaB and PCNA in p53-281G-expressing cells. However, on co-expression, p53 del 1-293 did not inhibit the tumorigenicity and colony forming ability of p53-281G expressing cells. Our earlier work showed that a deletion of the C-terminal sequences of p53-281G overlapping the oligomerization domain obliterates 'gain of function'. Taken together, the above information suggests that the C-terminal sequences have some critical role in 'gain of function' in addition to transactivation.

Activation of p53 protein by telomeric (TTAGGG)n repeats.

Genome instability is a primary factor leading to the activation of the p53 tumor suppressor protein. Telomeric repeat (TR) sequences are also responsible for genome integrity. By capping the termini of the chromosomes, TRs prevent them undergoing nucleolytic degradation, ligation or chromosome fusion. Interestingly, telomere shortening was suggested to activate p53, which in turn may cause primary cells to senesce. In order to elucidate the nature of a possible cross talk between the two, we introduced into cells TRs of defined length and investigated their effect on p53 activation and subsequent cellular response. We found that the introduction of a TR into cells leads to stabilization of the p53 protein. This stabilization was specific to TRs and was not observed in response to exposure of cells to plasmids containing non-TR sequences. p53 stabilization requires the presence of an intact p53 oligomerization domain. TR-activated p53 exhibited enhanced transcriptional activity. Eventually, TRs induced p53-dependent growth suppression, measured as a reduction in colony formation.

Identification of p53 sequence elements that are required for MDM2-mediated nuclear export.

It has been demonstrated that MDM2 can differentially regulate subcellular distribution of p53 and its close structural homologue p73. In contrast to MDM2-mediated p53 nuclear export, p73 accumulates in the nucleus as aggregates that colocalize with MDM2. Distinct distribution patterns of p53 and p73 suggest the existence of unique structural elements in the two homologues that determine their MDM2-mediated relocalization in the cell. Using a series of p53/p73 chimeric proteins, we demonstrate that three regions of p53 are involved in the regulation of MDM2-mediated nuclear export. The DNA binding domain (DBD) is involved in the maintenance of a proper conformation that is required for functional activity of the nuclear export sequence (NES) of p53. The extreme C terminus of p53 harbors several lysine residues whose ubiquitination by MDM2 appears to be the initial event in p53 nuclear export, as evidenced by the impaired nucleocytoplasmic shuttling of p53 mutants bearing simultaneous substitutions of lysines 370, 372, 373, 381, 382, and 386 to arginines (6KR) or alanines (6KA). Finally, the region between the DBD and the oligomerization domain of p53, specifically lysine 305, also plays a critical role in fully revealing p53NES. We conclude that MDM2-mediated nuclear export of p53 depends on a series of ubiquitination-induced conformational changes in the p53 molecule that lead to the activation of p53NES. In addition, we demonstrate that the p53NES may be activated without necessarily disrupting the p53 tetramer.