As species adapt to climatic changes, temperature-dependent functions of p53 in development, metabolism and cancer will adapt as well. Structural analyses of p53 epitopes interacting in response to environmental stressors, such as heat, may uncover physiologically relevant functions of p53 in cell regulation and genomic adaptations. Here we explore the multiple p53 elephant paradigm with an experimentally validated in silico model showing that under heat stress some p53 copies escape negative regulation by the MDM2 E3 ubiquitin ligase. Multiple p53 isoforms have evolved naturally in the elephant thus presenting a unique experimental system to study the scope of p53 functions and the contribution of environmental stressors to DNA damage. We assert that fundamental insights derived from studies of a historically heat-challenged mammal will provide important insights directly relevant to human biology in the light of climate change when 'heat' may introduce novel challenges to our bodies and health.

• Publication type
• Journal Article MeSH
• Review MeSH

BACKGROUND: The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated. METHODS: The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells. RESULTS: This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm. CONCLUSION: The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.

• Keywords
• DNA Damage Sensing, Genotoxic stress, MDM2, MRN complex, Precision medicine, RNA secondary structure, Synonymous mutations,
• MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 MeSH
• DNA Repair MeSH
• Polynucleotide 5'-Hydroxyl-Kinase * MeSH
• DNA Damage MeSH
• Proto-Oncogene Proteins c-mdm2 * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATM protein, human MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• Tumor Suppressor Protein p53 MeSH
• Polynucleotide 5'-Hydroxyl-Kinase * MeSH
• Proto-Oncogene Proteins c-mdm2 * MeSH

Cellular stress conditions activate p53-dependent pathways to counteract the inflicted damage. To achieve the required functional diversity, p53 is subjected to numerous post-translational modifications and the expression of isoforms. Little is yet known how p53 has evolved to respond to different stress pathways. The p53 isoform p53/47 (p47 or ΔNp53) is linked to aging and neural degeneration and is expressed in human cells via an alternative cap-independent translation initiation from the 2nd in-frame AUG at codon 40 (+118) during endoplasmic reticulum (ER) stress. Despite an AUG codon in the same location, the mouse p53 mRNA does not express the corresponding isoform in either human or mouse-derived cells. High-throughput in-cell RNA structure probing shows that p47 expression is attributed to PERK kinase-dependent structural alterations in the human p53 mRNA, independently of eIF2α. These structural changes do not take place in murine p53 mRNA. Surprisingly, PERK response elements required for the p47 expression are located downstream of the 2nd AUG. The data show that the human p53 mRNA has evolved to respond to PERK-mediated regulation of mRNA structures in order to control p47 expression. The findings highlight how p53 mRNA co-evolved with the function of the encoded protein to specify p53-activities under different cellular conditions.

• MeSH
• eIF-2 Kinase genetics   metabolism   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 * genetics   metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Protein Isoforms metabolism   MeSH
• Endoplasmic Reticulum Stress * genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• eIF-2 Kinase MeSH
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 * MeSH
• Protein Isoforms MeSH

The p53 tumor suppressor is a transcription factor with roles in cell development, apoptosis, oncogenesis, aging, and homeostasis in response to stresses and infections. p53 is tightly regulated by the MDM2 E3 ubiquitin ligase. The p53-MDM2 pathway has coevolved, with MDM2 remaining largely conserved, whereas the TP53 gene morphed into various isoforms. Studies on prevertebrate ancestral homologs revealed the transition from an environmentally induced mechanism activating p53 to a tightly regulated system involving cell signaling. The evolution of this mechanism depends on structural changes in the interacting protein motifs. Elephants such as Loxodonta africana constitute ideal models to investigate this coevolution as they are large and long-living as well as having 20 copies of TP53 isoformic sequences expressing a variety of BOX-I MDM2-binding motifs. Collectively, these isoforms would enhance sensitivity to cellular stresses, such as DNA damage, presumably accounting for strong cancer defenses and other adaptations favoring healthy aging. Here we investigate the molecular evolution of the p53-MDM2 system by combining in silico modeling and in vitro assays to explore structural and functional aspects of p53 isoforms retaining the MDM2 interaction, whereas forming distinct pools of cell signaling. The methodology used demonstrates, for the first time that in silico docking simulations can be used to explore functional aspects of elephant p53 isoforms. Our observations elucidate structural and mechanistic aspects of p53 regulation, facilitate understanding of complex cell signaling, and suggest testable hypotheses of p53 evolution referencing Peto's Paradox.

• Keywords
• Loxodonta africana, Peto’s Paradox, intrinsic specificity, lifespan, model, molecular evolution, p53 retrogenes, structural variations,
• MeSH
• Genes, p53 MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Neoplasms * genetics   MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   metabolism   MeSH
• Elephants * genetics   metabolism   MeSH
• Ubiquitination MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH
• Protein Isoforms MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH

Human cells are subjected to continuous challenges by different genotoxic stress attacks. DNA damage leads to erroneous mutations, which can alter the function of oncogenes or tumor suppressors, resulting in cancer development. To circumvent this, cells activate the DNA damage response (DDR), which mainly involves cell cycle regulation and DNA repair processes. The tumor suppressor p53 plays a pivotal role in the DDR by halting the cell cycle and facilitating the DNA repair processes. Various pathways and factors participating in the detection and repair of DNA have been described, including scores of RNA-binding proteins (RBPs) and RNAs. It has become increasingly clear that p53's role is multitasking, and p53 mRNA regulation plays a prominent part in the DDR. This review is aimed at covering the p53 RNA metabolism linked to the DDR and highlights the recent findings.

• Keywords
• ATM kinase, DNA damage response, MDM2, RNA metabolism, RNA-binding proteins, mRNA translation, p53,
• MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Mutation MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Untranslated Regions MeSH
• DNA Repair * physiology   MeSH
• DNA Damage * MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 MeSH
• Untranslated Regions MeSH
• RNA-Binding Proteins MeSH

BACKGROUND: The links between the p53/MDM2 pathway and the expression of pro-oncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1. METHODS: Proximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype. RESULTS: CD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control. CONCLUSION: Genetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

• Keywords
• Gene editing, MDM2, Nutlin-3, Protein-protein interactions, p53,
• MeSH
• B7 Antigens genetics   MeSH
• B7-H1 Antigen genetics   MeSH
• Cell Cycle drug effects   genetics   MeSH
• A549 Cells MeSH
• HCT116 Cells MeSH
• Imidazoles pharmacology   MeSH
• Humans MeSH
• Ligands MeSH
• Melanoma drug therapy   MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Piperazines pharmacology   MeSH
• Cell Proliferation drug effects   genetics   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   MeSH
• Up-Regulation drug effects   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• B7 Antigens MeSH
• B7-H1 Antigen MeSH
• CD274 protein, human MeSH Browser
• CD276 protein, human MeSH Browser
• Imidazoles MeSH
• Ligands MeSH
• MDM2 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• nutlin 3 MeSH Browser
• Piperazines MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH

Structured RNA regulatory motifs exist from the prebiotic stages of the RNA world to the more complex eukaryotic systems. In cases where a functional RNA structure is within the coding sequence a selective pressure drives a parallel co-evolution of the RNA structure and the encoded peptide domain. The p53-MDM2 axis, describing the interactions between the p53 tumor suppressor and the MDM2 E3 ubiquitin ligase, serves as particularly useful model revealing how secondary RNA structures have co-evolved along with corresponding interacting protein motifs, thus having an impact on protein - RNA and protein - protein interactions; and how such structures developed signal-dependent regulation in mammalian systems. The p53(BOX-I) RNA sequence binds the C-terminus of MDM2 and controls p53 synthesis while the encoded peptide domain binds MDM2 and controls p53 degradation. The BOX-I peptide domain is also located within p53 transcription activation domain. The folding of the p53 mRNA structure has evolved from temperature-regulated in pre-vertebrates to an ATM kinase signal-dependent pathway in mammalian cells. The protein - protein interaction evolved in vertebrates and became regulated by the same signaling pathway. At the same time the protein - RNA and protein - protein interactions evolved, the p53 trans-activation domain progressed to become integrated into a range of cellular pathways. We discuss how a single synonymous mutation in the BOX-1, the p53(L22 L), observed in a chronic lymphocyte leukaemia patient, prevents the activation of p53 following DNA damage. The concepts analysed and discussed in this review may serve as a conceptual mechanistic paradigm of the co-evolution and function of molecules having roles in cellular regulation, or the aetiology of genetic diseases and how synonymous mutations can affect the encoded protein.

• Keywords
• Ciona intestinalis, Intrinsically disordered proteins, Molecular basis of disease, Protein-RNA interactions, RNA world, Transcription factor, mRNA translation,
• MeSH
• Genetic Predisposition to Disease MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Tumor Suppressor Proteins genetics   metabolism   MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Neoplasms genetics   metabolism   pathology   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Gene Expression Regulation, Neoplastic * MeSH
• Gene Expression Profiling MeSH
• Transcriptome MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger MeSH
• Tumor Suppressor Proteins MeSH
• Tumor Suppressor Protein p53 MeSH
• RNA-Binding Proteins MeSH

A large number of signalling pathways converge on p53 to induce different cellular stress responses that aim to promote cell cycle arrest and repair or, if the damage is too severe, to induce irreversible senescence or apoptosis. The differentiation of p53 activity towards specific cellular outcomes is tightly regulated via a hierarchical order of post-translational modifications and regulated protein-protein interactions. The mechanisms governing these processes provide a model for how cells optimize the genetic information for maximal diversity. The p53 mRNA also plays a role in this process and this review aims to illustrate how protein and RNA interactions throughout the p53 mRNA in response to different signalling pathways control RNA stability, translation efficiency or alternative initiation of translation. We also describe how a p53 mRNA platform shows riboswitch-like features and controls the rate of p53 synthesis, protein stability and modifications of the nascent p53 protein. A single cancer-derived synonymous mutation disrupts the folding of this platform and prevents p53 activation following DNA damage. The role of the p53 mRNA as a target for signalling pathways illustrates how mRNA sequences have co-evolved with the function of the encoded protein and sheds new light on the information hidden within mRNAs.

• MeSH
• 3' Untranslated Regions genetics   MeSH
• 5' Untranslated Regions genetics   MeSH
• Stress, Physiological genetics   MeSH
• Humans MeSH
• Ligands MeSH
• RNA, Messenger genetics   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Proto-Oncogene Proteins c-mdm2 metabolism   MeSH
• Riboswitch genetics   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• 3' Untranslated Regions MeSH
• 5' Untranslated Regions MeSH
• Ligands MeSH
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• Riboswitch MeSH

p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

• Keywords
• ATM kinase, MDM2, cell signaling, intrinsically disordered proteins, p53 messenger RNA, synonymous mutations,
• MeSH
• Ataxia Telangiectasia Mutated Proteins genetics   metabolism   MeSH
• A549 Cells MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Phosphorylation genetics   physiology   MeSH
• Humans MeSH
• RNA, Small Interfering metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Mutation genetics   MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Polyribosomes metabolism   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   metabolism   MeSH
• Protein Stability MeSH
• Intrinsically Disordered Proteins genetics   metabolism   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ataxia Telangiectasia Mutated Proteins MeSH
• RNA, Small Interfering MeSH
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• TP53 protein, human MeSH Browser
• Intrinsically Disordered Proteins MeSH