Re-appraising the evidence for the source, regulation and function of p53-family isoforms.

The p53 family of proteins evolved from a common ancestor into three separate genes encoding proteins that act as transcription factors with distinct cellular roles. Isoforms of each member that lack specific regions or domains are suggested to result from alternative transcription start sites, alternative splicing or alternative translation initiation, and have the potential to exponentially increase the functional repertoire of each gene. However, evidence supporting the presence of individual protein variants at functional levels is often limited and is inferred by mRNA detection using highly sensitive amplification techniques. We provide a critical appraisal of the current evidence for the origins, expression, functions and regulation of p53-family isoforms. We conclude that despite the wealth of publications, several putative isoforms remain poorly established. Future research with improved technical approaches and the generation of isoform-specific protein detection reagents is required to establish the physiological relevance of p53-family isoforms in health and disease. In addition, our analyses suggest that p53-family variants evolved partly through convergent rather than divergent evolution from the ancestral gene.

Major histocompatibility class I antigenic peptides derived from translation of pre-mRNAs generate immune tolerance.

Antigenic peptides derived from introns are presented on major histocompatibility (MHC) class I molecules, but how these peptides are produced is poorly understood. Here, we show that an MHC class I epitope (SL8) sequence inserted in the second intron of the ß-globin gene in a C57BL/6 mouse (HBB) generates immune tolerance. Introduction of SL8-specific CD8+ T cells derived from OT-1 transgenic mice resulted in a threefold increase in OT-1 T cell proliferation in HBB animals, as compared to wild-type animals. The growth of MCA sarcoma cells expressing the intron-derived SL8 epitope was suppressed in wild-type animals compared to HBB mice. The ß-globin pre-mRNA was detected in the light polysomal fraction, and introducing stop codons identified a non-AUG initiation site between +228 and +255 nts upstream of the SL8. Isolation of ribosome footprints confirmed translation initiation within this 27 nt sequence. Furthermore, treatment with splicing inhibitor shifts the translation of the pre-mRNA to monosomal fractions and results in an increase of intron-derived peptide substrate as shown by polysome profiling and cell imaging. These results show that non-AUG-initiated translation of pre-mRNAs generates peptides for MHC class I immune tolerance and helps explain why alternative tissue-specific splicing is tolerated by the immune system.

The DNA damage sensor ATM kinase interacts with the p53 mRNA and guides the DNA damage response pathway.

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.

Type I arginine methyltransferases are intervention points to unveil the oncogenic Epstein-Barr virus to the immune system.

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome maintenance but is also highly antigenic. Hence, EBV seemingly evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 limits the translation of its own mRNA to the minimal level to ensure its essential function, thereby, at the same time, minimizing immune recognition. Therefore, defining intervention points at which to interfere with GAr-based inhibition of translation is an important step to trigger an immune response against EBV-carrying cancers. The host protein nucleolin (NCL) plays a critical role in this process via a direct interaction with G-quadruplexes (G4) formed in the GAr-encoding sequence of the viral EBNA1 mRNA. Here we show that the C-terminal arginine-glycine-rich (RGG) motif of NCL is crucial for its role in GAr-based inhibition of translation by mediating interaction of NCL with G4 of EBNA1 mRNA. We also show that this interaction depends on the type I arginine methyltransferase family, notably PRMT1 and PRMT3: drugs or small interfering RNA that target these enzymes prevent efficient binding of NCL on G4 of EBNA1 mRNA and relieve GAr-based inhibition of translation and of antigen presentation. Hence, this work defines type I arginine methyltransferases as therapeutic targets to interfere with EBNA1 and EBV immune evasion.

The nascent polypeptide-associated complex (NAC) controls translation initiation in cis by recruiting nucleolin to the encoding mRNA.

Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine-alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence.

The Elephant Evolved p53 Isoforms that Escape MDM2-Mediated Repression and Cancer.

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.

Reduced murine double minute 2 and 4 protein, but not messenger RNA, expression is associated with more severe disease in myelodysplastic syndromes and acute myeloblastic leukaemia.

The human homologues of murine double minute 2 (MDM2) and 4 (MDM4) negatively regulate p53 tumour suppressor activity and are reported to be frequently overexpressed in human malignancies, prompting clinical trials with drugs that prevent interactions between MDM2/MDM4 and p53. Bone marrow samples from 111 patients with acute myeloblastic leukaemia, myelodysplastic syndrome or chronic myelomonocytic leukaemia were examined for protein (fluorescence-activated cell sorting) and messenger RNA (mRNA) expression (quantitative polymerase chain reaction) of MDM2, MDM4 and tumour protein p53 (TP53). Low protein expression of MDM2 and MDM4 was observed in immature cells from patients with excess of marrow blasts (>5%) compared with CD34+ /CD45low cells from healthy donors and patients without excess of marrow blasts (<5%). The mRNA levels were indistinguishable in all samples examined regardless of disease status or blast levels. Low MDM2 and MDM4 protein expression were correlated with poor survival. These data show a poor correlation between mRNA and protein expression levels, suggesting that quantitative flow cytometry analysis of protein expression levels should be used to predict and validate the efficacy of MDM2 and MDM4 inhibitors. These findings show that advanced disease is associated with reduced MDM2 and MDM4 protein expression and indicate that the utility of MDM2 and MDM4 inhibitors may have to be reconsidered in the treatment of advanced myeloid malignancies.

Substrate-specific presentation of MHC class I-restricted antigens via autophagy pathway.

The accumulation of protein aggregates is toxic and linked to different diseases such as neurodegenerative disorders, but the role of the immune system to target and destroy aggregate-carrying cells is still relatively unknown. Here we show a substrate-specific presentation of antigenic peptides to the direct MHC class I pathway via autophagy. We observed no difference in presentation of peptides derived from the viral EBNA1 protein following suppression of autophagy by knocking down Atg5 and Atg12. However, the same knock down treatment suppressed the presentation from ovalbumin. Fusing the aggregate-prone poly-glutamine (PolyQ) to the ovalbumin had no effect on antigen presentation via autophagy. Interestingly, fusing the EBNA1-derived gly-ala repeat (GAr) sequence to ovalbumin rendered the presentation Atg5/12 independent. We also demonstrate that the relative levels of protein expression did not affect autophagy-mediated antigen presentation. These data suggest a substrate-dependent presentation of antigenic peptides for the MHC class I pathway via autophagy and indicate that the GAr of the EBNA1 illustrates a novel virus-mediated mechanism for immune evasion of autophagy-dependent antigen presentation.

What do we need to know and understand about p53 to improve its clinical value?

Few proteins are more studied than the p53 tumour suppressor, but what have we learned from these studies and what do we really know about p53 that can benefit clinical practice? The DNA sequence encoding p53 is frequently mutated in cancers but the functional outcomes of single mutations, in respect to loss or gain of different activities, especially in relation to immune evasion, are not clear. This illustrates p53's complexity which even after 40 years keeps providing surprises, but also explains why it has not yet lived up to its potential to benefit cancer treatment. We have reassessed a few key experiments that shaped the p53 field and we take a closer look at the interpretations of these experiments: what they have taught us, the resulting dogmas, and their potential clinical importance. One outcome is a more dynamic view of p53 in terms of its activity, its regulation, and downstream effectors, which will benefit the clinical application of p53 for diagnosis, prognosis, and therapy. Mutations and regulatory factors can have different effects on p53 activity depending on context, important but neglected aspects when interpreting p53 and its pathways in cancers. Even though p53 is undoubtedly unique as a multifunctional hub in different cellular pathways, the concept of a factor taking up different functions within a regulatory pathway during different conditions is not. In this sense, p53 continues to lead the way for a better understanding of the cellular and molecular mechanisms underlying cancer development in vivo. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

HDMX folds the nascent p53 mRNA following activation by the ATM kinase.

Regulated protein synthesis via changes in mRNA structures forms an important part of how prokaryotic cells adapt protein expression in response to changes in the environment. Little is known regarding how this concept has adapted to regulate mRNA translation via signaling pathways in mammalian cells. Here, we show that following phosphorylation by the ataxia telangiectasia mutated (ATM) kinase at serine 403, the C-terminal RING domain of HDMX binds the nascent p53 mRNA to promote a conformation that supports the p53 mRNA-HDM2 interaction and the induction of p53 synthesis. HDMX and its homolog HDM2 bind the same p53 internal ribosome entry sequences (IRES) structure but with different specificity and function. The results show how HDMX and HDM2 act as nonredundant IRES trans-acting factors (ITAFs) to bring a positive synergistic effect on p53 expression during genotoxic stress by first altering the structure of the newly synthesized p53 mRNA followed by stimulation of translation.

Hydrogen deuterium exchange mass spectrometry identifies the dominant paratope in CD20 antigen binding to the NCD1.2 monoclonal antibody.

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.

MDM2's dual mRNA binding domains co-ordinate its oncogenic and tumour suppressor activities.

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.

Low potential of circulating interleukin 1 receptor antagonist as a prediction marker for squamous cell carcinoma of the head and neck.

BACKGROUND: Circulating markers are attractive molecules for prognosis and management of cancer that allow sequential monitoring of patients during and after treatment. Based on previous protein profiling data, circulating interleukin 1 receptor antagonist (IL-1Ra) was evaluated as a potential diagnostic and prognostic marker for squamous cell carcinomas of the head and neck (SCCHN). In this study, we aimed at confirming the clinical relevance of plasma IL-1Ra in SCCHN and exploring its potential as a prediction marker for SCCHN. METHODS: Plasma from 87 patients with SCCHN, control plasma from 28 healthy individuals and pre-diagnostic plasma from 44 patients with squamous cell carcinoma of the oral tongue (SCCOT) and 88 matched controls were analysed with IL-1Ra electrochemiluminescence immunoassays from mesoscale diagnostics. RESULTS: Plasma IL-1Ra was found to be up-regulated in patients with oral tongue, gingiva and base of tongue tumours compared to healthy individuals (p < 0.01). IL-1Ra levels positively correlated with tumour size (p < 0.01) and body mass index (p = 0.013). Comparing pre-diagnostic plasma to the matched controls, similar IL1-Ra levels were seen (p = 0.05). CONCLUSION: The anti-inflammatory cytokine IL-1Ra could be a diagnostic marker for SCCHN, whereas its potential as a cancer prediction marker was not supported by our data.

Resistance mechanisms to inhibitors of p53-MDM2 interactions in cancer therapy: can we overcome them?

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.

The p53 mRNA: an integral part of the cellular stress response.

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.

Nuclear processing of nascent transcripts determines synthesis of full-length proteins and antigenic peptides.

Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.

Molecular Determinants and Specificity of mRNA with Alternatively-Spliced UPF1 Isoforms, Influenced by an Insertion in the 'Regulatory Loop'.

The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the 'regulatory loop (involved in mRNA binding)'. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: 'open (isoform_2; without insertion)' and 'closed (isoform_1; with insertion)'. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the 'open conformation'. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA-UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein-mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.

PD-L1 in squamous cell carcinoma of the oral tongue shows gender-specific association with prognosis.

OBJECTIVE: To use alternative quantitation approaches to clarify the clinical implication of programmed cell death ligand 1 (PD-L1) in squamous cell carcinoma of the oral tongue (SCCOT). MATERIALS AND METHODS: Ventana SP263 immunohistochemistry assay and a multiplicative QuickScore method were applied to quantify PD-L1 in tumor and surrounding immune cells from 101 patients with SCCOT. Tumor-infiltrating immune cells were estimated from bulk tissue transcriptional profiles of 25 patients. Circulating PD-L1 levels were measured in serum from 30 patients using an electrochemiluminescence assay platform. RESULTS: We found higher tumor cell PD-L1 levels in females than males (p = .019). For patients with low PD-L1 in tumor cells, better survival was seen in males than females (overall survival p = .021, disease-free survival p = .020). Tumor-infiltrating natural killer T cells, immature dendritic cells, and M1 macrophages were positively associated with tumor cell PD-L1 (p < .05). CONCLUSIONS: Our data confirmed the significance of gender on tumor cell PD-L1 expression and demonstrated combined effects of gender and PD-L1 levels on clinical outcome in patients with SCCOT. The data also indicated the involvement of specific immune cell types in PD-L1-regulated immune evasion.

Allosteric changes in HDM2 by the ATM phosphomimetic S395D mutation: implications on HDM2 function.

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.