P53 DNA Binding Domain Research Articles

IASPP mediates p53 selectivity through a modular mechanism fine-tuning DNA recognition

The most frequently mutated protein in human cancer is p53, a transcription factor (TF) that regulates myriad genes instrumental in diverse cellular outcomes including growth arrest and cell death. Cell context-dependent p53 modulation is critical for this life-or-death balance, yet remains incompletely understood. Here we identify sequence signatures enriched in genomic p53-binding sites modulated by the transcription cofactor iASPP. Moreover, our p53-iASPP crystal structure reveals that iASPP displaces the p53 L1 loop-which mediates sequence-specific interactions with the signature-corresponding base-without perturbing other DNA-recognizing modules of the p53 DNA-binding domain. A TF commonly uses multiple structural modules to recognize its cognate DNA, and thus this mechanism of a cofactor fine-tuning TF-DNA interactions through targeting a particular module is likely widespread. Previously, all tumor suppressors and oncoproteins that associate with the p53 DNA-binding domain-except the oncogenic E6 from human papillomaviruses (HPVs)-structurally cluster at the DNA-binding site of p53, complicating drug design. By contrast, iASPP inhibits p53 through a distinct surface overlapping the E6 footprint, opening prospects for p53-targeting precision medicine to improve cancer therapy.

Analysis of the TP53 Deleterious Single Nucleotide Polymorphisms Impact on Estrogen Receptor Alpha-p53 Interaction: A Machine Learning Approach.

Breast cancer is a leading cancer type and one of the major health issues faced by women around the world. Some of its major risk factors include body mass index, hormone replacement therapy, family history and germline mutations. Of these risk factors, estrogen levels play a crucial role. Among the estrogen receptors, estrogen receptor alpha (ERα) is known to interact with tumor suppressor protein p53 directly thereby repressing its function. Previously, we have studied the impact of deleterious breast cancer-associated non-synonymous single nucleotide polymorphisms (nsnps) rs11540654 (R110P), rs17849781 (P278A) and rs28934874 (P151T) in TP53 gene on the p53 DNA-binding core domain. In the present study, we aimed to analyze the impact of these mutations on p53–ERα interaction. To this end, we, have modelled the full-length structure of human p53 and validated its quality using PROCHECK and subjected it to energy minimization using NOMAD-Ref web server. Three-dimensional structure of ERα activation function-2 (AF-2) domain was downloaded from the protein data bank. Interactions between the modelled native and mutant (R110P, P278A, P151T) p53 with ERα was studied using ZDOCK. Machine learning predictions on the interactions were performed using Weka software. Results from the protein–protein docking showed that the atoms, residues and solvent accessibility surface area (SASA) at the interface was increased in both p53 and ERα for R110P mutation compared to the native complexes indicating that the mutation R110P has more impact on the p53–ERα interaction compared to the other two mutants. Mutations P151T and P278A, on the other hand, showed a large deviation from the native p53-ERα complex in atoms and residues at the surface. Further, results from artificial neural network analysis showed that these structural features are important for predicting the impact of these three mutations on p53–ERα interaction. Overall, these three mutations showed a large deviation in total SASA in both p53 and ERα. In conclusion, results from our study will be crucial in making the decisions for hormone-based therapies against breast cancer.

Probing direct interaction of oncomiR-21-3p with the tumor suppressor p53 by fluorescence, FRET and atomic force spectroscopy

miRNA-21-3p is overexpressed in a number of cancers and contributes to their development with a concomitant inhibition of the p53 onco-suppressive function. While a direct interaction of p53 with some miRNA precursors (namely pri-miRNAs and pre-miRNAs) was found, no interaction with mature micro RNA has been so far evidenced. It could therefore be very interesting to investigate if a direct interaction of miR-21-3p and p53 is occurring with possible impairment of the p53 onco-suppressive function. Fluorescence and Atomic Force Spectroscopy (AFS) were applied to study the interaction of p53 DNA Binding Domain (DBD) and miRNA-21-3p. Förster resonance energy transfer (FRET) was used to measure the distance between the DBD lone tryptophan (FRET donor) and a dye (FRET acceptor) bound to miRNA-21-3p. AFS and Fluorescence evidenced a direct interaction between miRNA-21-3p and DBD; with the formed complex being characterized by an affinity of 105 M, with a lifetime in the order of seconds. FRET allowed to determine an average distance of 4.0 nm between the DBD lone Trp146 and miRNA-21-3p; consistently with the involvement of the DBD L3 loop and/or the H1 helix in the complex formation, directly involved in the oligomerization and DNA binding. This may suggest that a functional inhibition of p53 could arise from its interaction with the oncogenic miRNA. Evidence of DBD-miRNA-21-3p complex formation may deserve some interest for inspiring novel therapeutic strategies.

Loss of the p53 transactivation domain results in high amyloid aggregation of the Δ40p53 isoform in endometrial carcinoma cells

Dysfunctional p53 formation and activity can result from aberrant expression and subcellular localization of distinct p53 isoforms or aggregates. Endometrial carcinoma (EC) is a cancer type in which p53 status is correlated with prognosis, and TP53 mutations are a frequent genetic modification. Here we aimed to evaluate the expression patterns of different p53 isoforms and their contributions to the formation and subcellular localization of p53 amyloid aggregates in both EC and endometrial nontumor cell lines. We found that full-length (fl) p53 and a truncated p53 isoform, Δ40p53, resulting from alternative splicing of exon 2 or alternative initiation of translation at ATG-40, are the predominantly expressed p53 variants in EC cells. However, Δ40p53 was the major p53 isoform in endometrial nontumor cells. Immunofluorescence assays revealed that Δ40p53 is mainly localized to cytoplasmic punctate structures of EC cells, resembling solid-phase structures similar to those found in neurodegenerative pathologies. Using light-scattering kinetics, CD, and transmission EM, we noted that the p53 N-terminal transactivation domain significantly reduces aggregation of the WT p53 DNA-binding domain, confirming the higher aggregation tendency of Δ40p53, which lacks this domain. This is the first report of cytoplasmic Δ40p53 in EC cells being a major component of amyloid aggregates. The differential aggregation properties of p53 isoforms in EC cells may open up new avenues in the development of therapeutic strategies that preferentially target specific p53 isoforms to prevent p53 amyloid aggregate formation.

P53 Binds Preferentially to Non-B DNA Structures Formed by the Pyrimidine-Rich Strands of GAA·TTC Trinucleotide Repeats Associated with Friedreich's Ataxia.

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich’s ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich’s ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs’ non-B DNA structures.

Hsp70- and Hsp90-Mediated Regulation of the Conformation of p53 DNA Binding Domain and p53 Cancer Variants.

The activity of the tumor suppressor p53 has to be timed and balanced closely to prevent untimely induction of cell death. The stability of p53 depends on the ubiquitin ligase Mdm2 but also on Hsp70 and Hsp90 chaperones that interact with its DNA binding domain (DBD). Using hydrogen exchange mass spectrometry and biochemical methods, we analyzed conformational states of wild-type p53-DBD at physiological temperatures and conformational perturbations in three frequent p53 cancer mutants. We demonstrate that the Hsp70/Hdj1 system shifts the conformational equilibrium of p53 toward a flexible, more mutant-like, DNA binding inactive state by binding to the DNA binding loop. The analyzed cancer mutantsare likewise destabilized by interaction with the Hsp70/Hdj1 system. In contrast, Hsp90 protects the DBD of p53 wild-type and mutant proteins from unfolding. We propose that the Hsp70 and Hsp90 chaperone systems assume complementary functions to optimally balance conformational plasticity with conformational stability.

748 Duplex sequencing reveals the effects of caffeine on reducing UV-induced mutations of cancer-relevant genes

Epidemiological studies have shown that daily caffeinated coffee intake is associated with decreased risks of developing skin cancer. Importantly, this cancer-preventive effect is attributable to caffeine (no benefit with decaffeinated coffee) and is dose-dependent. However, the mechanism by which caffeine prevents skin cancer remains elusive. To determine the effects of caffeine on chronic UV-induced genetic mutations and skin cancer development, we irradiated Xpc–/– (DNA repair-deficient) mice with 25–250 mJ/cm2 of UVB per day, twice a week for 19 weeks and treated with or without caffeine topically immediately after each UV irradiation. Chronic UV irradiation to mouse back skin caused cutaneous squamous cell carcinoma (cSCC). Topical application of caffeine significantly inhibited cSCC development compared to vehicle treatment (n = 70 mice per group). We collected UV-irradiated but nontumorous skin from these mice for Duplex Sequencing, a next-generation sequencing method capable of detecting ultralow-frequency mutations. Among seven genes analyzed (Trp53, Cdkn2a, Tert, Sftpc, Ambp, B2m, and Cyc1), the most prevalent mutation in UV-irradiated skin was the UV signature C>T mutation. C>T mutation on the noncoding strand was essentially absent in transcriptionally active genes, likely due to transcription-coupled repair that removes UV-induced DNA lesions only on the transcribed (noncoding) strand. Caffeine did not drastically change the overall mutation frequency in UV-irradiated skin. Instead, caffeine reduced the frequency of mutations in the p53 DNA-binding domain and the p16/Cdkn2a promoter. These findings indicate mechanisms by which caffeine may have selectively sensitized p53-mutant or p16-deficient keratinocytes to death in vivo, a general concept that has been suggested in prior studies performed in cell lines in vitro.

C-terminal α Domain of p63 Binds to p300 to Coactivate β-Catenin

TP63 (p63), a member of the tumor suppressor TP53 (p53) gene family, is essential for ectodermal tissue development and suppresses malignant progression of carcinomas. The most abundant isoform, ΔNp63α (referred to as p63), lacks the N-terminal transactivation (TA) domain, and was originally characterized as a dominant-negative type suppressor against p53 family proteins. It also binds to TCF/LEF to inhibit β-catenin. Nevertheless, transcriptional activation by p63 has also been observed in varied systems. To understand the puzzling results, we analyzed the structure–function relationship of p63 in the control of β-catenin-dependent transcription. p63 acted as a suppressor of moderately induced β-catenin. However, when nuclear targeted S33Y β-catenin was applied to cause the maximum enhancer activation, p63 displayed a β-catenin-coactivating function. The DNA-binding domain of p63 and the target sequence facilitated it. Importantly, we newly found that, despite the absence of TA domain, p63 was associated with p300, a general adaptor protein and chromatin modifier causing transcriptional activation. C-terminal α domain of p63 was essential for p300-binding and for the coactivator function. These results were related to endogenous p63-p300 complex formation and Wnt/β-catenin-responsive gene regulation by p63 in squamous cell carcinoma lines. The novel p63-p300 interaction may be involved in positive regulation of gene expression in tissue development and carcinogenesis.

Simulations of mutant p53 DNA binding domains reveal a novel druggable pocket.

The DNA binding domain (DBD) of the tumor suppressor p53 is the site of several oncogenic mutations. A subset of these mutations lowers the unfolding temperature of the DBD. Unfolding leads to the exposure of a hydrophobic β-strand and nucleates aggregation which results in pathologies through loss of function and dominant negative/gain of function effects. Inspired by the hypothesis that structural changes that are associated with events initiating unfolding in DBD are likely to present opportunities for inhibition, we investigate the dynamics of the wild type (WT) and some aggregating mutants through extensive all atom explicit solvent MD simulations. Simulations reveal differential conformational sampling between the WT and the mutants of a turn region (S6–S7) that is contiguous to a known aggregation-prone region (APR). The conformational properties of the S6–S7 turn appear to be modulated by a network of interacting residues. We speculate that changes that take place in this network as a result of the mutational stress result in the events that destabilize the DBD and initiate unfolding. These perturbations also result in the emergence of a novel pocket that appears to have druggable characteristics. FDA approved drugs are computationally screened against this pocket.

Mutant p63 Affects Epidermal Cell Identity through Rewiring the Enhancer Landscape

Transcription factor p63 is a key regulator of epidermal keratinocyte proliferation and differentiation. Mutations in the p63 DNA-binding domain are associated with ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome. However, the underlying molecular mechanism of these mutations remains unclear. Here, we characterized the transcriptome and epigenome of p63 mutant keratinocytes derived from EEC patients. The transcriptome of p63 mutant keratinocytes deviated from the normal epidermal cell identity. Epigenomic analyses showed an altered enhancer landscape in p63 mutant keratinocytes contributed by loss of p63-bound active enhancers and unexpected gain of enhancers. The gained enhancers were frequently bound by deregulated transcription factors such as RUNX1. Reversing RUNX1 overexpression partially rescued deregulated gene expression and the altered enhancer landscape. Our findings identify a disease mechanism whereby mutant p63 rewires the enhancer landscape and affects epidermal cell identity, consolidating the pivotal role of p63 in controlling the enhancer landscape of epidermal keratinocytes.

Interaction of the anticancer p28 peptide with p53-DBD as studied by fluorescence, FRET, docking and MD simulations

BackgroundThe p28 peptide, derived from the blue copper protein Azurin, exerts an anticancer action due to interaction with the tumor suppressor p53, likely interfering with its down-regulators. Knowledge of both the kinetics and topological details of the interaction, could greatly help to understand the peptide anticancer mechanism. MethodsFluorescence and Förster resonance energy transfer (FRET) were used to determine both the binding affinity and the distance between the lone tryptophan (FRET donor) of DNA Binding Domain (DBD) of p53 and the Iaedens dye (FRET acceptor) bound to the p28 peptide. Docking, Molecular Dynamic simulations and free energy binding calculations were used to single out the best complex model, compatible with the distance measured by FRET. ResultsTryptophan fluorescence quenching provided a 105 M−1 binding affinity for the complex. Both FRET donor fluorescence quenching and acceptor enhancement are consistent with a donor-acceptor distance of about 2.6 nm. Docking and molecular dynamics simulations allowed us to select the best complex, enlightening the contact regions between p28 and DBD. Conclusionsp28 binds to DBD partially engaging the L1 loop, at the same region of the p53 down-regulator COP1, leaving however the DNA binding site available for functional interactions. General significanceElucidation of the DBD-p28 complex gets insights into the functional role of p28 in regulating the p53 anticancer activity, also offering new perspectives to design new drugs able to protect the p53 anticancer function.

C16-ceramide is a natural regulatory ligand of p53 in cellular stress response

Ceramides are important participants of signal transduction, regulating fundamental cellular processes. Here we report the mechanism for activation of p53 tumor suppressor by C16-ceramide. C16-ceramide tightly binds within the p53 DNA-binding domain (Kd ~ 60 nM), in close vicinity to the Box V motif. This interaction is highly selective toward the ceramide acyl chain length with its C10 atom being proximal to Ser240 and Ser241. Ceramide binding stabilizes p53 and disrupts its complex with E3 ligase MDM2 leading to the p53 accumulation, nuclear translocation and activation of the downstream targets. This mechanism of p53 activation is fundamentally different from the canonical p53 regulation through protein–protein interactions or posttranslational modifications. The discovered mechanism is triggered by serum or folate deprivation implicating it in the cellular response to nutrient/metabolic stress. Our study establishes C16-ceramide as a natural small molecule activating p53 through the direct binding.

Abstract 556: Oncogenic properties and response to HDAC inhibitor treatment of H1299 cells expressing GOF p53 mutants

Abstract The tumor suppressor p53 protein is mutated in over 50% of cancers. The most frequent mutations in p53, termed “hotspot” mutations, occur in the DNA binding domain of the p53 protein. These mutations not only remove normal p53 function but also convey additional oncogenic properties greater than loss of tumor suppressor activities associated with loss of wild-type (WT) p53 activities. With newly acquired oncogenic properties, mutations in the DNA binding domain of p53 are classified as gain of function (GOF) mutations. GOF p53 mutants can be subdivided into conformational mutations (e.g. R175H, H193Y) involving residues necessary for maintenance of structure and contact mutations (e.g. R248Q, R273H) involving resides necessary for DNA interaction. Cancer cells bearing a GOF p53 mutation increase metastasis with application of histone deacetylase inhibitors (HDACi). This uncharacterized pro-oncogenic GOF p53 property is of clinical relevance as HDACi are FDA approved for hematological cancers but have failed in clinical trials for solid tumors. To explore this hypothesis, metastatic capacity and proliferation rates of common GOF p53 mutations were assessed with and without HDACi treatment and in the presence or absence of specific inhibitors of target GOF p53. The p53-null, H1299 non-small cell lung carcinoma line was transfected with GOF p53 mutants and WT p53 using a tetracycline inducible expression system. Inducible expression of stable subclones was varified by immunofluorescence and western blot. Proliferation rate was measured by incorporation of EdU and metastatic capacity was assessed by measuring MMP activity and migration assays with and without HDACi treatment. Specific GOF p53 mutations possess a subset of the oncogenic properties of the entire GOF p53 class. Data suggest that the GOF p53 mutants display differing rates of proliferation with the R175H mutation exhibiting the highest rate. Application of NSC319726 significantly decreases the proliferation rate and appears to successfully refold the R175H mutant to a WT conformation. The R248Q mutation exhibited morphological change suggestive of the epithelial to mesenchymal transition associated with increased metastatic capacity. Preliminary data suggest that HDACi affect among these cell lines in conjunction with NSC 319726 and SCH 529074 treatment. In conclusion, HDACi treatment may be contraindicated while treatment with HDACi may have great benefit in tumors which are WT or null for p53 protein expression. Citation Format: Brianna Flores, Elizabeth E. Hull. Oncogenic properties and response to HDAC inhibitor treatment of H1299 cells expressing GOF p53 mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 556.

In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy.

Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA-binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53-MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of p53-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3α and MI773 efficiently inhibited the p53-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein-protein interactions and evaluation of inhibitors in living cells.

The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding.

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.

Cloning the P53 DNA Binding Domain

P53 is a tumor suppressor protein that binds to the DNA and is responsible for regulating cell growth. Mutations of p53 are associated with numerous types of cancers in patients worldwide. Mutants of p53 affect not only its structure, but its original function, causing p53 to gain oncogenic functions. The focus of this research is to clone the DNA binding domain (DBD) from the wild type p53 as well as its most common mutants and to characterize their p53 DBD. It is proposed that when missense mutations are present within the p53 DBD, p53 will destabilize, leading to its aggregation within the cell. We used molecular cloning techniques such as PCR to isolate the DBD from p53. We also utilized mammalian cell viability assays such as MTT and cytotoxicity to determine the effects of p53 on human cells. PCR results showed that DBD was amplified from full length p53 and cloned into expression vectors pET28 and pcDNA3.1. In our preliminary results both the MTT and cytotoxicity assays showed that WT p53 decreases the viability of mammalian cells. Therefore, we successfully cloned the DBD from p53 and are in the process of characterizing the effects of mutant p53 on cell viability and aggregation propensity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism of An Anticancer Peptide Rescuing p53 from Degradation by COP1

A 28 amino‐acid peptide derived from azurin (amino acids 50–77, p28) preferentially enters human cancer cells and induces apoptosis or growth inhibition. Previous studies have revealed that p28 exerts its anticancer activity mainly by interacting with the p53 DNA binding domain (DBD) and interfering in the p53 degradation signaling pathway. However, the interacting mechanisms between p28 and p53‐DBD are missing and how p28 inhibits p53‐DBD ubiquitination remains largely unknown. In this work, we characterized the interacting mechanisms between p28 and p53‐DBD by a combination of NMR, X‐ray crystallography, ITC and computer simulations. Interactions between p28 and p53‐DBD were mainly mediated by hydrophobic contacts. Structural models of the p28/p53‐DBD complex obtained with NMR datadriven docking indicated that p28 adopted an α‐helix upon binding to p53‐DBD. Val59, Val60, Thr61, Tyr72 and Leu73 of p28 made extensive interactions with the L1 loop and S7–S8 loop of p53‐DBD. Site‐directed mutagenesis and ITC characterization showed that single alanine substitution of the above mentioned hydrophobic residues reduced the binding affinity by more than one order of magnitude. GST pull‐down assays showed that the interactions between p53‐ DBD and the E3 ligase COP1 were blocked by p28. Since alanine substitution of key hydrophobic residues reduced the binding affinity of p28 with p53‐DBD, alanine mutated p28 were less efficient in blocking the p53‐DBD/COP1 interaction. Determination of the p28/p53‐ DBD complex structure is in progress, which will allow us to rationally design p28 derived peptides exhibiting higher affinity with p53‐DBD and stronger anticancer activity in the future.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (Grant number: 21603121, Y.H.) and Hubei University of Technology (M. G. and Y.H.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MDMX acidic domain inhibits p53 DNA binding in vivo and regulates tumorigenesis

The MDM2 homolog MDMX oncoprotein is indispensable for inhibition of p53 during normal embryonic development and malignant transformation, yet how MDMX harnesses p53 functions is unclear. In addition to a canonical N-terminal p53-binding domain, recent work suggests the central acidic domain of MDMX regulates p53 interaction through intramolecular mimicry and engages in second-site interaction with the p53 core domain in vitro. To test the physiological relevance of these interactions, we generated an MDMX knockin mouse having substitutions in a conserved WW motif necessary for these functions (W201S/W202G). Notably, MDMXSG cells have normal p53 level but increased p53 DNA binding and target gene expression, and rapidly senesce. In vivo, MDMXSG inhibits early-phase disease in Eµ-Myc transgenic mice but accelerates the onset of lethal lymphoma and shortens overall survival. Therefore, MDMX is an important regulator of p53 DNA binding, which complements the role of MDM2 in regulating p53 level. Furthermore, the results suggest that the WW motif has dual functions that regulate p53 and inhibit Myc-driven lymphomas independent of p53.

Deciphering the Interactions between an Anticancer Bacteriocin and the P53 DNA Binding Domain

Deciphering the Interactions between an Anticancer Bacteriocin and the P53 DNA Binding Domain

Characterization of an Hsp90-Independent Interaction between Co-Chaperone p23 and Transcription Factor p53.

Cancer-suppressing transcription factor p53 is regulated by a wide variety of cellular factors, including many chaperones. The DNA-binding domain (DBD) of p53 is known to interact with the chaperone Hsp90, but the role of other members of the chaperone network, including co-chaperones such as p23, is unknown. Using a combination of nuclear magnetic resonance (NMR) titration, isothermal titration calorimetry, fluorescence anisotropy, and native agarose gel electrophoresis, we have identified a direct interaction between the p53 DBD and Hsp90 co-chaperone p23 that occurs in the absence of Hsp90. The affinity is relatively weak and largely determined by electrostatic interactions between the acidic C-terminal disordered tail of p23 and the two DNA-binding regions of the p53 DBD. We show by NMR and native agarose gel electrophoresis that a p53-specific double-stranded DNA sequence competes successfully with p23 for binding to the p53 DBD. The Hsp90 independence of the interaction between p23 and p53 DBD, together with the competition of p23 versus DNA for p53, raises the intriguing possibility that p23, like other small charged proteins, may affect p53 in hitherto unknown ways.