P53 Tetramer Research Articles

The human ΔNp53 isoform triggers metabolic and gene expression changes that activate mTOR and alter mitochondrial function

A naturally occurring p53 isoform that lacks 39 residues at the N-terminus (denoted ΔNp53), when expressed with wild-type p53 (WTp53), forms mixed ΔNp53:WTp53 tetramers and causes accelerated aging in mice. Cellular alterations specific to ΔNp53:WTp53 have been difficult to assess because ΔNp53 and WTp53 coexpression results in tetramer heterogeneity, including formation of contaminating WTp53 tetramers. Based on the p53 tetramer structure, we expressed ΔNp53 and WTp53 as a single transcript that maintained tetramer architecture, ensuring a 2:2 ΔNp53:WTp53 stoichiometry. As expected, ΔNp53:WTp53 tetramers were stable and transcriptionally active in vitro and in cells, largely mimicking the function of WTp53 tetramers. Microarray analyses, however, revealed about 80 genes whose expression was altered twofold or more in ΔNp53:WTp53 cells. Moreover, global metabolomic profiling quantitated hundreds of biochemicals across different experiments (WTp53, ΔNp53:WTp53, plus controls). When evaluated collectively, these data suggested altered mTOR signaling and mitochondrial function-each canonical regulators of longevity-in cells expressing ΔNp53:WTp53 vs. WTp53. Increased levels of free amino acids, increased expression of IRS-1, and decreased expression of INPP5D/SHIP-1 suggested activated mTOR signaling in ΔNp53:WTp53 cells; this was confirmed upon comparative analyses of several mTOR pathway intermediates. We also observed changes in mitochondrial function in ΔNp53:WTp53 cells, which correlated with increased MARS2 expression and increased levels of carnitine, acetyl CoA, ATP, and Krebs cycle intermediates. Finally, increased levels of succinate and 2-hydroxyglutarate indicate potential epigenetic means to propagate ΔNp53:WTp53-induced gene expression changes to cell progeny. This may be especially important for aging, as biological effects manifest over time.

RhoGAPs Attenuate Cell Proliferation by Direct Interaction with p53 Tetramerization Domain

Many Rho GTPase activation proteins (RhoGAPs) are deleted or downregulated in cancers, but the functional consequences are still unclear. Here, we show that the RhoGAP ArhGAP11A induces cell-cycle arrest and apoptosis by binding to the tumor suppressor p53. The RhoGAP domain of ArhGAP11A binds to the tetramerization domain of p53, but not to its family members p63 or p73. The interaction stabilizes the tetrameric conformation of p53 and enhances its DNA-binding activity, thereby inducing cell-cycle arrest and apoptosis. Upon DNA damage stress, ArhGAP11A accumulates in the nucleus andinteracts with p53, whereas knockdown of ArhGAP11A partially blocks p53 transcriptional activity. These findings explain why RhoGAPs are frequently deleted in cancers and suggest that the RhoGAP family sits at the crossroads between the cell-migration and proliferation pathways.

P53 DNA Binding Cooperativity Is Essential for Apoptosis and Tumor Suppression In Vivo

Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.

Abstract 568: Analysis of p53 oligomerization and sensitivity to checkpoint abrogation of neuroblastoma.

Abstract Cell cycle checkpoints prevent cells from dividing with damaged DNA by causing cell cycle arrest or apoptosis. DNA damage-induced cell cycle arrest resulting from treatment with the topoisomerase I inhibitor SN38 can be abrogated specifically in p53-defective cells by treatment with the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). However, some p53 wild-type tumor cell lines have also been shown to be sensitive to checkpoint abrogation, suggesting that these cells have other defects in the p53 pathway that can be exploited for therapeutic purposes. Our recent studies revealed that cells that are sensitive to checkpoint abrogation lack p53 dimers and tetramers. Thus, one possible explanation for the sensitivity of p53 wildtype tumors to checkpoint abrogation is a defect in oligomerization. In the current study, we investigate the DNA damage response and UCN-01 sensitivity of three p53 wildtype neuroblastoma cell lines: SK-N-SH, SH-SY5Y, and IMR32. In order to determine the responses of these cells to DNA damage, the cells were treated with concentrations of SN38 ranging from 0-30 ng/ml. All three cell lines arrested in G2, S, or G1 depending upon SN38 concentration and displayed an increase in p53 levels with increasing SN38. Additionally, p53 was phosphorylated on serine 15 following SN38 treatment in all three cell lines, suggesting that p53 is active. In order to determine whether these cells are susceptible to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 3 ng/ml SN38 for 24 hours, followed by 25-100 nM UCN-01 for 6 and 24 hours. The SK-N-SH and IMR32 showed only slight sensitivity to the highest concentration of UCN-01 treatment as evidenced by partial abrogation of S and G2 arrest. The SH-SY5Y, however, abrogated S arrest after 6 hours at the lowest concentration of UCN-01 and abrogated G2 arrest after 24 hours. We also analyzed the oligomerization status of p53 using glutaraldehyde crosslinking. The SK-N-SH cells possess levels of p53 dimers and tetramers similar to what has previously been reported in p53 wildtype MCF10A cells. The IMR32 and SH-SY5Y, however, had extremely low levels of dimers and tetramers. Consistent with this, only SK-N-SH showed activation of p21waf1 in response to SN38 treatment. Previous studies have reported cytoplasmic sequestration as a mechanism of p53 inactivation p53 wildtype neuroblastomas, although more recent studies refute these reports. In order to determine the sub-cellular distribution of p53, we prepared nuclear and cytoplasmic extracts. Both the SK-N-SH and SH-SY5Y had primarily nuclear p53, while in IMR32, p53 was evenly distributed between the nucleus and cytoplasm. The results of this study suggest that oligomerization status alone is insufficient as an indicator of sensitivity of p53 wildtype tumors to the therapeutic combination of DNA damage agent and checkpoint inhibitor. Citation Format: Robert Lipski, Aime A. Levesque. Analysis of p53 oligomerization and sensitivity to checkpoint abrogation of neuroblastoma. [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 568. doi:10.1158/1538-7445.AM2013-568

Crystal Structures of the DNA-binding Domain Tetramer of the p53 Tumor Suppressor Family Member p73 Bound to Different Full-site Response Elements

How cells choose between developmental pathways remains a fundamental biological question. In the case of the p53 protein family, its three transcription factors (p73, p63, and p53) each trigger a gene expression pattern that leads to specific cellular pathways. At the same time, these transcription factors recognize the same response element (RE) consensus sequences, and their transactivation of target genes overlaps. We aimed to understand target gene selectivity at the molecular level by determining the crystal structures of the p73 DNA-binding domain (DBD) in complex with full-site REs that vary in sequence. We report two structures of the p73 DBD bound as a tetramer to 20-bp full-site REs based on two distinct quarter-sites: GAACA and GAACC. Our study confirms that the DNA-binding residues are conserved within the p53 family, whereas the dimerization and tetramerization interfaces diverge. Moreover, a conserved lysine residue in loop L1 of the DBD senses the presence of guanines in positions 2 and 3 of the quarter-site RE, whereas a conserved arginine in loop 3 adapts to changes in position 5. Sequence variations in the RE elicit a p73 conformational response that might explain target gene specificity.

Potential Conformational Heterogeneity of p53 Bound to S100B(ββ)

The negative regulatory domain (NRD) of the p53 tumor suppressor is intrinsically disordered. It contains several posttranslational modification (PTM) sites that are important for regulation of p53 activity. Calcium-dependent binding of dimeric S100B(ββ) to p53-NRD blocks access to these PTM sites and disrupts the p53 tetramer to inhibit p53 activation. Previous nuclear magnetic resonance (NMR) structural studies have suggested that p53-NRD folds into a stable helix upon binding to S100B(ββ). Intriguingly, despite the well-converged and stably folded nature of the NMR structure ensemble, experimentally resolved intermolecular nuclear Overhauser enhancements (NOEs) are extremely weak; most have 5- to 6-Å upper bounds, and mainly involve the C-terminal segment of p53-NRD. Such a systematic lack of strong intermolecular NOEs could suggest that the p53/S100B(ββ) interface is more dynamic than currently believed. Indeed, extensive atomistic simulations in explicit solvent (with 1.0μs total effective sampling) revealed large heterogeneity in the S100B(ββ)-bound conformation of p53-NRD. Helix unwinding at the C-terminus allows key hydrophobic residues (Leu383 and Phe385) to make more extensive intermolecular contacts, whereas the highly helical N-terminus displays substantial flexibility in packing with S100B(ββ). Importantly, the predicted heterogeneous ensemble as a whole is highly consistent with experimental intermolecular NOEs, although many conformational sub-states coexist and individual sub-states satisfy only subsets of the NOE restraints. Furthermore, the simulated ensemble provides similar shielding of key PTM sites to support p53 inhibition. This study not only provides new insights into the structural basis of the p53/S100B(ββ) recognition but also highlights the importance of recognizing dynamic complexes in structural studies of intrinsically disordered protein interactions.

62 Distinct nucleosome organization around p53 response elements associated with cell cycle arrest and apoptosis

DNA is severely deformed in a tetrameric complex with the tumor suppressor protein p53, both in solution and in co-crystal structures. The DNA deformations occur not only at the CWWG motifs in the p53 half sites, but also in the central region of a full site, YYRR. As a result, the DNA fragment is bent in such a way that the p53 tetramer is located on the external side of the DNA loop. The overall shape of the p53-bound DNA (bending and sliding) resembles nucleosomal DNA, suggesting that p53 may recognize its cognate site embedded in the nucleosome. Recent studies established unambiguously that p53 is a nucleosome-binding protein. In particular, the p53 site in a nucleosome is accessible if it is bent in the direction similar to that found in the p53-DNA co-crystals; the site becomes inaccessible if the orientation is changed by ∼180° (see Figure). This implies that the rotational setting of a p53 site in nucleosomes is critical for its accessibility, which may have a direct impact on how p53 recognizes its target binding sites in the chromatin context. We illustrate the functional importance of this idea by comparing the p53 sites associated with cell cycle arrest (CCA-sites) and sites associated with apoptosis (Apo-sites), the two extreme cellular outcomes after the p53 activation. To elucidate the rotational setting of p53 sites in nucleosomal DNA, we developed a computational approach (Cui & Zhurkin, 2010) based on well-established DNA sequence patterns related to nucleosome positioning. Importantly, we found that the CCA-sites are oriented in such a way that they tend to be ‘open’ and ‘exposed’ on the nucleosomal surface. This result is corroborated by human nucleosomes mapped in high resolution (Gaffney et al., 2012). The nucleosome dyad positions are “out of phase” with the CCA-sites, separated by ∼10n + 5 bp from the site centers. In other words, the CCA-sites embedded in nucleosomes are accessible for direct p53 recognition, thereby facilitating p53 binding and subsequent gene induction. By contrast, our computations suggest that most of the Apo-sites have a different rotational setting from the CCA-sites, consistent with the high-resolution nucleosome mapping. That is, the Apo-sites in general are likely to be ‘closed’ in the chromatin context. This would hinder direct p53 binding to DNA, and require additional factors such as chromatin remodeling complexes to expose the Apo-sites, which may account for the ‘delayed’ kinetics of apoptotic genes in vivo.

Deformation of a P53 Response Element Revealed by Site-Directed Spin Labeling

The tumor suppressor protein p53 is called the “guardian of the genome” and has been implicated in a growing number of biological processes, including cell cycle arrest, senescence, apoptosis, autophagy, metabolism, and aging. As a transcription factor, p53 regulates a large number of signaling pathways by interacting with a diverse set of DNAs, called p53 response elements (REs), which are composed of two decameric consensus half-sites separated by a spacer. It has been proposed that DNA conformation changes upon p53 binding, which may vary between different REs, play important roles in p53-DNA recognition. However, details on such DNA conformational changes are limited, as currently there is no high-resolution structure of unbound RE, although a number of crystal structures of tetrameric p53 fragments bound to artificial or naturally occurring REs have been reported. Here site-directed spin labeling is used to probe solution conformations of an RE regulating protein p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation. Using a nucleotide-independent nitroxide probe and Double-Electron-Electron Resonance spectroscopy, nanometer distances between various DNA sites were measured in unbound p21RE as well as in that complexed to the p53 DNA binding domain. Models of the unbound p21RE, which were selected using the measured distances from a large pool generated by Monte-Carlo simulations, reveal major conformational changes at the half-site interface as compared to the bound DNA reported in a crystal structure. These results shed light on the mechanism of DNA recognition by p53.

The Regulatory Domain Stabilizes the p53 Tetramer by Intersubunit Contacts with the DNA Binding Domain

The tumor suppressor protein p53 is often referred to as the guardian of the genome. In the past, controversial findings have been presented for the role of the C-terminal regulatory domain (RD) of p53 as both a negative regulator and a positive regulator of p53 activity. However, the underlying mechanism remained enigmatic. To understand the function of the RD and of a dominant phosphorylation site within the RD, we analyzed p53 variants in vivo and in vitro. Our experiments revealed, surprisingly, that the p53 RD of one subunit interacts with the DNA binding domain of an adjacent subunit in the tetramer. This leads to the formation of intersubunit contacts that stabilize the tetrameric state of p53 and enhance its transcriptional activity in a cooperative manner. These effects are further modulated by phosphorylation of a conserved serine within the RD.

The Tetramer of p53 in the Absence of DNA Forms a Relaxed Quaternary State

p53 is a tetrameric multidomain protein that triggers the anticancer cellular response to stress. We have calculated a three-dimensional reconstruction of full-length human p53 in the absence of DNA using single-particle electron microscopy. The reconstruction of DNA-free full-length p53 shows a square-shaped structure with four distinct domains and a hollow center. In comparison with the known compacted DNA-bound full-length p53 structures, the DNA-free p53 tetramer adopts a relaxed conformation with separated monomers and oligomerization interfaces different from those of the DNA-bound conformation.

Sequence-dependent sliding kinetics of p53

Proper timing of gene expression requires that transcription factors (TFs) efficiently locate and bind their target sites within a genome. Theoretical studies have long proposed that one-dimensional sliding along DNA while simultaneously reading its sequence can accelerate TF's location of target sites. Sliding by prokaryotic and eukaryotic TFs were subsequently observed. More recent theoretical investigations have argued that simultaneous reading and sliding is not possible for TFs without their possessing at least two DNA-binding modes. The tumor suppressor p53 has been shown to slide on DNA, and recent experiments have offered structural and single molecule support for a two-mode model for the protein. If the model is applicable to p53, then the requirement that TFs be able to read while sliding implies that noncognate sites will affect p53's mobility on DNA, which will thus be generally sequence-dependent. Here, we confirm this prediction with single-molecule microscopy measurements of p53's local diffusivity on noncognate DNA. We show how a two-mode model accurately predicts the variation in local diffusivity, while a single-mode model does not. We further determine that the best model of sequence-specific binding energy includes terms for "hemi-specific" binding, with one dimer of tetrameric p53 binding specifically to a half-site and the other binding nonspecifically to noncognate DNA. Our work provides evidence that the recognition by p53 of its targets and the timing thereof can depend on its noncognate binding properties and its ability to change between multiple modes of binding, in addition to the much better-studied effects of cognate-site binding.

Domain–domain interactions in full-length p53 and a specific DNA complex probed by methyl NMR spectroscopy

The tumor suppressor p53 is a homotetramer of 4 × 393 residues. Its core domain and tetramerization domain are linked and flanked by intrinsically disordered sequences, which hinder its full structural characterization. There is an outstanding problem of the state of the tetramerization domain. Structural studies on the isolated tetramerization domain show it is in a folded tetrameric conformation, but there are conflicting models from electron microscopy of the full-length protein, one of which proposes that the domain is not tetramerically folded and the tetrameric protein is stabilized by interactions between the N and C termini. Here, we present methyl-transverse relaxation optimized NMR spectroscopy (methyl-TROSY) investigations on the full-length and separate domains of the protein with its methionine residues enriched with (13)C to probe its quaternary structure. We obtained high-quality spectra of both the full-length tetrameric p53 and its DNA complex, observing the environment at 11 specific methyl sites. The tetramerization domain was as tetramerically folded in the full-length constructs as in the isolated domain. The N and C termini were intrinsically disordered in both the full-length protein and its complex with a 20-residue specific DNA sequence. Additionally, we detected in the interface of the core (DNA-binding) and N-terminal parts of the protein a slow conformational exchange process that was modulated by specific recognition of DNA, indicating allosteric processes.

P53 Searches on DNA by Rotation-Uncoupled Sliding at C-Terminal Tails and Restricted Hopping of Core Domains

The tumor suppressor p53 is a transcription factor that searches its cognate sites on DNA. During the search, the roles and interplay of its two DNA binding domains, the folded core domain and the disordered C-terminal domain (CTD), have been controversial. Here, we performed molecular simulations of p53 at various salt concentrations finding that, at physiological salt concentration, p53 diffuses along nonspecific DNA via rotation-uncoupled sliding with its CTD, whereas the core domain repeats dissociation and association. This is in perfect agreement with a recent single molecule experiment. In the simulation of tetrameric full-length p53, two DNA binding domains both bound to nonspecific DNA in a characteristic form at low salt concentration, whereas at physiological salt concentration, only CTD kept bound to DNA and the core domain frequently hopped on DNA. Simulations of a construct that lacks the core domain (TetCD) clarified rotation-uncoupled diffusion on nonspecific DNA. At low salt concentration, the diffusion constant due to sliding was dependent on the salt concentration, which differs from the prediction of a classic theory of transcription factors. At physiological salt concentration, it was independent of the salt concentration, in harmony with experiments. Moreover, we found that the sliding via the CTD follows the helical pitch of DNA (i.e., rotation-coupled sliding) at low salt concentration while it is virtually uncoupled to the helical pitch, a hallmark of rotation-uncoupled sliding at physiological salt concentration.

P53 Dimers Associate with a Head-to-Tail Response Element to Repress Cyclin B Transcription

DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.

Specific recognition of p53 tetramers by peptides derived from p53 interacting proteins.

Oligomerization plays a major role in regulating the activity of many proteins, and in modulating their interactions. p53 is a homotetrameric transcription factor that has a pivotal role in tumor suppression. Its tetramerization domain is contained within its C-terminal domain, which is a site for numerous protein-protein interactions. Those can either depend on or regulate p53 oligomerization. Here we screened an array of peptides derived from proteins known to bind the tetrameric p53 C-terminal domain (p53CTD) and identified ten binding peptides. We quantitatively characterized their binding to p53CTD using fluorescence anisotropy. The peptides bound tetrameric p53CTD with micromolar affinities. Despite the high charge of the binding peptides, electrostatics contributed only mildly to the interactions. NMR studies indicated that the peptides bound p53CTD at defined sites. The most significant chemical shift deviations were observed for the peptides WS100B(81–92), which bound directly to the p53 tetramerization domain, and PKCα(281–295), which stabilized p53CTD in circular dichroism thermal denaturation studies. Using analytical ultracentrifugation, we found that several of the peptides bound preferentially to p53 tetramers. Our results indicate that the protein-protein interactions of p53 are dependent on the oligomerization state of p53. We conclude that peptides may be used to regulate the oligomerization of p53.

Abstract 2220: p53 dimers associate with a head-to-head element to repress cyclin B transcription

Abstract DNA damage activates cell cycle checkpoints to promote cell cycle arrest. p53 is a major player in checkpoints by activating expression of the cdk inhibitor p21 and repressing expression of cyclin B. DNA damage-induced S and G2 arrest following treatment with the topoisomerase I inhibitor SN38 is abrogated in p53-defective cells by treatment with the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wildtype MCF10A cells with derivatives whose p53 function is inhibited by overexpression of the tetramerization domain (MCF10A/OD) or overexpression of shRNA against p53 (MCF10A/αp53) (Levesque et al, Oncogene 24, 3786, 2005). Treatment of MCF10A/OD with SN38 followed by UCN-01 results in abrogation of S, but not G2, arrest, while the MCF10A/αp53 abrogated both S and G2 arrest. The S abrogation correlated with reduced p21, consistent with the requirement of p53 tetramerization for activation of p21 expression. Failure of G2 abrogation correlated with reduced cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B expression. In the present study, we confirmed that the p53-dependent repression of cyclin B in response to DNA damage occurs at the transcriptional level by analyzing cyclin B mRNA levels. Treatment of MCF10A and MCF10A/OD with SN38 resulted in reduction of cyclin B mRNA levels whereas treatment of MCF10A/αp53 with SN38 did not. This reflects the change in protein levels following SN38 treatment. We next analyzed p53 oligomerization using glutaraldehyde crosslinking. Following SN38 treatment, MCF10A contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. MCF10A/OD, however, possessed monomers and dimers, but lacked trimers and tetramers. These results suggest that p53 monomers or dimers are involved in repression of cyclin B. One model to explain the p53-mediated repression of cyclin B in MCF10A/OD is that p53 dimers, rather than tetramers, bind to a p53 response element in the cyclin B promoter. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-tail orientation, the cyclin B promoter contains similar elements oriented head-to-head. We performed both chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSA) to determine whether p53 binds to this head-to-head element. ChIP assays revealed that p53 associates with this element in MCF10A and MCF10A/OD but not in MCF10A/αp53. We performed EMSA using a biotin-labeled probe containing the head-to-head element and observed shifts in mobility consistent with the molecular weight of tetramers and dimers using MCF10A nuclear extract (NE), but observed only the dimeric shift with MCF10A/OD NE, and no shift with MCF10A/αp53 NE. Taken together, these results suggest p53 dimers associate with the head-to-head element to repress cyclin B transcription. 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 2220. doi:1538-7445.AM2012-2220

Abstract 1176: Bypassing the dominant-negative effect of mutant p53 in cancer cells

Abstract The tumor suppressor p53 is mutated in more than 50% of all cancers. The transcriptional activity of p53 depends on localization to the nucleus and formation of p53 tetramers. Use of wild-type (wt) p53 for gene therapy is limited due to the dominant-negative effect of mutant (mut) p53 over wt p53. Mut p53 will hetero-oligomerize with wt p53 via the tetramerization domain (TD) found in p53, deactivating the tumor suppressor function of wt p53. In order to use p53 for gene therapy, an alternative oligomerization domain for p53 was investigated. The coiled-coil (CC) domain from Bcr (breakpoint cluster region), similar to the native TD of p53, can form an antiparallel dimer of dimers. The TD from p53 was then substituted with this CC, and the activity of this new p53 (p53-CC) was tested. We evaluated the transcriptional activity of p53-CC compared to wt p53 in a dual luciferase reporter gene assay. To demonstrate the ability of our p53-CC to bypass the dominant-negative effect prompted by hetero-oligomers, the reporter gene assay was carried in human breast adenocarcinoma (MCF-7) and human breast ductal carcinoma (T47D) cell lines. MCF-7 cells contain wt p53 population that is mislocalized to the cytoplasm, whereas, T47D cells contain mutant p53. Indeed, data from the reporter gene experiment shows a reduction in wt p53 activity introduced to T47D cells compared to MCF-7 cells, while the activity of p53-CC remains consistent regardless of the presence of an endogenous mut p53 population. We also tested the biological activity of p53-CC and its ability to induce apoptosis using 7-AAD and Annexin-V assays (late stage apoptosis). In addition, Caspase-3/7 assay (mid-stage apoptosis) was performed to further validate a p53-dependent apoptotic pathway. Data obtained from the aforementioned experiments suggest that p53-CC has a very similar biological activity to wt p53. Furthermore, we examined the gene expression profile of p53-CC using wt p53 as a reference. The assay was performed using real-time PCR array technology to analyze the expression of 84 genes related to p53-mediated signal transduction. Two negative controls were used; the first is a p53 construct that lacks its tetramerization domain (p53ΔTDC) and should not possess any transcriptional activity, and the second is the CC by itself. Indeed, p53-CC shows comparable gene expression profile to wt p53 and has the ability to transactivate the various p53 target genes. In summary, p53-CC retains p53 activity and excludes hetero-oligomer formation, overcoming a major barrier in using p53 for cancer gene therapy. 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 1176. doi:1538-7445.AM2012-1176

Inhibition of tumor suppressor protein p53-dependent transcription by a tetramerization domain peptide via hetero-oligomerization

Tumor suppressor protein p53 induces cell cycle arrest, apoptosis, and senescence in response to cellular stresses. The p53 tetramer formation is essential for its functions. Despite of these crucial functions of p53 for integrity of genome, activation of the p53 signal pathway causes low induced pluripotent stem (iPS) cell generation efficiency. In this study, we report transient inhibition of p53-dependent transcription using a p53 tetramerization domain peptide that contains cell penetrating and nuclear localization signals. The peptide was efficiently introduced into cells and inhibited p21 expression via hetero-tetramerization with endogenous p53 protein. This method can be applied towards safe and efficient iPS cell generation.

Crystal Structure of a Multidomain Human p53 Tetramer Bound to the Natural CDKN1A (p21) p53-Response Element

The p53 tumor suppressor protein is a sequence-specific DNA-binding transcription factor. Structures of p53 bound to DNA have been described, but, so far, no structure has been determined of p53 bound to a natural p53-response element. We describe here the structure of a human p53 homotetramer encompassing both the DNA-binding and homo-oligomerization domains in complex with the natural p53-response element present upstream of the promoter of the CDKN1A (p21) gene. Similar to our previously described structures of human p53 tetramers bound to an artificial consensus DNA site, p53 DNA binding proceeds via an induced fit mechanism with loops L1 of two subunits adopting recessed conformations. Interestingly, the conformational change involving loop L1 is even more extreme than the one previously observed with the artificial consensus DNA site. In fact, the previously determined loop L1 conformation seems to be a transition intermediate between the non-DNA-bound and CDKN1A-bound states. Thus, the new structure further supports our model that recognition of specific DNA by p53 is associated with conformational changes within the DNA-binding domain of p53.

Structure and Kinetic Stability of the p63 Tetramerization Domain

The p53 family of transcription factors—comprising p53, p63 and p73—plays an important role in tumor prevention and development. Essential to their function is the formation of tetramers, allowing cooperative binding to their DNA response elements. We solved crystal structures of the human p63 tetramerization domain, showing that p63 forms a dimer of dimers with D2 symmetry composed of highly intertwined monomers. The primary dimers are formed via an intramolecular β-sheet and hydrophobic helix packing (H1), a hallmark of all p53 family members. Like p73, but unlike p53, p63 requires a second helix (H2) to stabilize the architecture of the tetramer. In order to investigate the impact of structural differences on tetramer stability, we measured the subunit exchange reaction of p53 family homotetramers by nanoflow electrospray mass spectrometry. There were differences in both the kinetics and the pattern of the exchange reaction, with the p53 and p63 tetramers exhibiting much faster exchange kinetics than p73. The structural similarity between p63 and p73 rationalizes previous observations that p63 and p73 form mixed tetramers, and the kinetic data reveal the dissociation of the p73 homotetramers as the rate-limiting step for heterotetramer formation. Differential stability of the tetramers may play an important role in the cross talk between different isoforms and regulation of p53, p63 and p73 function in the cell cycle.