HDMX and its homologue HDM2 are two essential proteins for the cell; after genotoxic stress, both are phosphorylated near to their RING domain, specifically at serine 403 and 395, respectively. Once phosphorylated, both can bind the p53 mRNA and enhance its translation; however, both recognize p53 protein and provoke its degradation under normal conditions. HDM2 has been well-recognized as an E3 ubiquitin ligase, whereas it has been reported that even with the high similarity between the RING domains of the two homologs, HDMX does not have the E3 ligase activity. Despite this, HDMX is needed for the proper p53 poly-ubiquitination. Phosphorylation at serine 395 changes the conformation of HDM2, helping to explain the switch in its activity, but no information on HDMX has been published. Here, we study the conformation of HDMX and its phospho-mimetic mutant S403D, investigate its E3 ligase activity and dissect its binding with p53. We show that phospho-mutation does not change the conformation of the protein, but HDMX is indeed an E3 ubiquitin ligase in vitro; however, in vivo, no activity was found. We speculated that HDMX is regulated by induced fit, being able to switch activity accordingly to the specific partner as p53 protein, p53 mRNA or HDM2. Our results aim to contribute to the elucidation of the contribution of the HDMX to p53 regulation.

• Keywords
• HDM2, HDMX, Induced fit, MDM2, MDMX, cancer, p53, ubiquitination,
• MeSH
• Nuclear Proteins genetics   MeSH
• RNA, Messenger metabolism   MeSH
• Tumor Suppressor Protein p53 * genetics   metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Proto-Oncogene Proteins c-mdm2 * genetics   metabolism   MeSH
• Proto-Oncogene Proteins genetics   MeSH
• Serine metabolism   MeSH
• Ubiquitin genetics   MeSH
• Ubiquitination MeSH
• Ubiquitin-Protein Ligases genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nuclear Proteins MeSH
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 * MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 * MeSH
• Proto-Oncogene Proteins MeSH
• Serine MeSH
• Ubiquitin MeSH
• Ubiquitin-Protein Ligases MeSH

Since the discovery of the first MDM2 inhibitors, we have gained deeper insights into the cellular roles of MDM2 and p53. In this review, we focus on MDM2 inhibitors that bind to the p53-binding domain of MDM2 and aim to disrupt the binding of MDM2 to p53. We describe the basic mechanism of action of these MDM2 inhibitors, such as nutlin-3a, summarise the determinants of sensitivity to MDM2 inhibition from p53-dependent and p53-independent points of view and discuss the problems with innate and acquired resistance to MDM2 inhibition. Despite progress in MDM2 inhibitor design and ongoing clinical trials, their broad use in cancer treatment is not fulfilling expectations in heterogenous human cancers. We assess the MDM2 inhibitor types in clinical trials and provide an overview of possible sources of resistance to MDM2 inhibition, underlining the need for patient stratification based on these aspects to gain better clinical responses, including the use of combination therapies for personalised medicine.

• Keywords
• Combination therapy, MDM2, MDM2 inhibitor, Nutlin-3a, Personalised medicine, Resistance, p53,
• MeSH
• Drug Resistance, Bacterial drug effects   physiology   MeSH
• Molecular Targeted Therapy methods   MeSH
• Clinical Trials as Topic MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 antagonists & inhibitors   genetics   metabolism   MeSH
• Neoplasms drug therapy   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Proto-Oncogene Proteins c-mdm2 antagonists & inhibitors   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• MDM2 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Antineoplastic Agents MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• TP53 protein, human MeSH Browser

The p53 and Mouse double minute 2 (MDM2) proteins are hubs in extensive networks of interactions with multiple partners and functions. Intrinsically disordered regions help to adopt function-specific structural conformations in response to ligand binding and post-translational modifications. Different techniques have been used to dissect interactions of the p53-MDM2 pathway, in vitro, in vivo, and in situ each having its own advantages and disadvantages. This review uses the p53-MDM2 to show how different techniques can be employed, illustrating how a combination of in vitro and in vivo techniques is highly recommended to study the spatio-temporal location and dynamics of interactions, and to address their regulation mechanisms and functions. By using well-established techniques in combination with more recent advances, it is possible to rapidly decipher complex mechanisms, such as the p53 regulatory pathway, and to demonstrate how protein and nucleotide ligands in combination with post-translational modifications, result in inter-allosteric and intra-allosteric interactions that govern the activity of the protein complexes and their specific roles in oncogenesis. This promotes elegant therapeutic strategies that exploit protein dynamics to target specific interactions.

• Keywords
• ATM *, DNA damage response *, MDM2 *, MDMX *, p53 *, p53 mRNA *, post-translational modification *, protein-RNA interactions *, protein-protein interactions *,
• MeSH
• Phosphorylation genetics   MeSH
• Nuclear Proteins MeSH
• Humans MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• DNA Damage genetics   MeSH
• Protein Processing, Post-Translational genetics   MeSH
• Cell Cycle Proteins genetics   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   MeSH
• Protein Binding 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
• Review MeSH
• Names of Substances
• Nuclear Proteins MeSH
• MDM2 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 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

Cell growth requires a high level of protein synthesis and oncogenic pathways stimulate cell proliferation and ribosome biogenesis. Less is known about how cells respond to dysfunctional mRNA translation and how this feeds back into growth regulatory pathways. The Epstein-Barr virus (EBV)-encoded EBNA1 causes mRNA translation stress in cis that activates PI3Kδ. This leads to the stabilization of MDM2, induces MDM2's binding to the E2F1 mRNA and promotes E2F1 translation. The MDM2 serine 166 regulates the interaction with the E2F1 mRNA and deletion of MDM2 C-terminal RING domain results in a constitutive E2F1 mRNA binding. Phosphorylation on serine 395 following DNA damage instead regulates p53 mRNA binding to its RING domain and prevents the E2F1 mRNA interaction. The p14Arf tumour suppressor binds MDM2 and in addition to preventing degradation of the p53 protein it also prevents the E2F1 mRNA interaction. The data illustrate how two MDM2 domains selectively bind specific mRNAs in response to cellular conditions to promote, or suppress, cell growth and how p14Arf coordinates MDM2's activity towards p53 and E2F1. The data also show how EBV via EBNA1-induced mRNA translation stress targets the E2F1 and the MDM2 - p53 pathway.

• MeSH
• Cell Cycle genetics   MeSH
• Phosphorylation genetics   MeSH
• Carcinogenesis genetics   MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Tumor Suppressor Protein p14ARF genetics   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Neoplasms genetics   virology   MeSH
• Oncogenes genetics   MeSH
• DNA Damage genetics   MeSH
• Cell Proliferation genetics   MeSH
• Protein Domains genetics   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   MeSH
• RNA Recognition Motif Proteins genetics   MeSH
• E2F1 Transcription Factor genetics   MeSH
• Genes, Tumor Suppressor MeSH
• Herpesvirus 4, Human genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• E2F1 protein, human MeSH Browser
• MDM2 protein, human MeSH Browser
• RNA, Messenger MeSH
• Tumor Suppressor Protein p14ARF MeSH
• Tumor Suppressor Protein p53 MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• RNA Recognition Motif Proteins MeSH
• TP53 protein, human MeSH Browser
• E2F1 Transcription Factor MeSH

Allosteric changes imposed by post-translational modifications regulate and differentiate the functions of proteins with intrinsic disorder regions. HDM2 is a hub protein with a large interactome and with different cellular functions. It is best known for its regulation of the p53 tumour suppressor. Under normal cellular conditions, HDM2 ubiquitinates and degrades p53 by the 26S proteasome but after DNA damage, HDM2 switches from a negative to a positive regulator of p53 by binding to p53 mRNA to promote translation of the p53 mRNA. This change in activity is governed by the ataxia telangiectasia mutated kinase via phosphorylation on serine 395 and is mimicked by the S395D phosphomimetic mutant. Here we have used different approaches to show that this event is accompanied by a specific change in the HDM2 structure that affects the HDM2 interactome, such as the N-termini HDM2-p53 protein-protein interaction. These data will give a better understanding of how HDM2 switches from a negative to a positive regulator of p53 and gain new insights into the control of the HDM2 structure and its interactome under different cellular conditions and help identify interphases as potential targets for new drug developments.

• Keywords
• ATM, DNA damage, MDM2, p53, phosphomimetic mutation,
• MeSH
• Allosteric Regulation MeSH
• Amino Acid Motifs MeSH
• Ataxia Telangiectasia Mutated Proteins genetics   metabolism   MeSH
• Phosphorylation MeSH
• Humans MeSH
• Mutation, Missense * MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-mdm2 chemistry   genetics   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ataxia Telangiectasia Mutated Proteins MeSH
• MDM2 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• TP53 protein, human MeSH Browser

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