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.

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.

The epithelial to mesenchymal transition (EMT) is a cellular program that drives de-differentiation of cells in both physiological and pathological processes. One of the characteristics of cells describing an EMT is the (re)acquisition of a motility capacity that allows them to migrate through the original tissue as well as to other sites in the organism. The molecular mechanisms that control the EMT are rapidly emerging and here we add to the idea that the adaptation required for cells to commit to the EMT includes adjustments of the translation machinery and metabolic pathways to cope with a high demand of extracellular components.

Cellular tRNAs appear today as a diverse population of informative macromolecules with conserved general elements ensuring essential common functions and different and distinctive features securing specific interactions and activities. Their differential expression and the variety of post-transcriptional modifications they are subject to, lead to the existence of complex repertoires of tRNA populations adjusted to defined cellular states. Despite the tRNA-coding genes redundancy in prokaryote and eukaryote genomes, it is surprising to note the absence of genes coding specific translational-active isoacceptors throughout the phylogeny. Through the analysis of different releases of tRNA databases, this review aims to provide a general summary about those "missing tRNA genes." This absence refers to both tRNAs that are not encoded in the genome, as well as others that show critical sequence variations that would prevent their activity as canonical translation adaptor molecules. Notably, while a group of genes are universally missing, others are absent in particular kingdoms. Functional information available allows to hypothesize that the exclusion of isodecoding molecules would be linked to: 1) reduce ambiguities of signals that define the specificity of the interactions in which the tRNAs are involved; 2) ensure the adaptation of the translational apparatus to the cellular state; 3) divert particular tRNA variants from ribosomal protein synthesis to other cellular functions. This leads to consider the "missing tRNA genes" as a source of putative non-canonical tRNA functions and to broaden the concept of adapter molecules in ribosomal-dependent protein synthesis.

Hepatocellular carcinoma (HCC) usually afflicts individuals in their maturity after a protracted liver disease. Contrasting with this pattern, the age structure of HCC in Andean people displays a bimodal distribution with half of the patients developing HCC in adolescence and early adulthood. To deepen our understanding of the molecular determinants of the disease in this population, we conducted an integrative analysis of gene expression and DNA methylation in HCC developed by 74 Peruvian patients, including 39 adolescents and young adults. While genome-wide hypomethylation is considered as a paradigm in human HCCs, our analysis revealed that Peruvian tumors are associated with a global DNA hypermethylation. Moreover, pathway enrichment analysis of transcriptome data characterized an original combination of signatures. Peruvian HCC forgoes canonical activations of IGF2, Notch, Ras/MAPK, and TGF-β signals to depend instead on Hippo/YAP1, MYC, and Wnt/β-catenin pathways. These signatures delineate a homogeneous subtype of liver tumors at the interface of the proliferative and non-proliferative classes of HCCs. Remarkably, the development of this HCC subtype occurs in patients with one of the four Native American mitochondrial haplogroups A-D. Finally, integrative characterization revealed that Peruvian HCC is apparently controlled by the PRC2 complex that mediates cell reprogramming with massive DNA methylation modulating gene expression and pinpointed retinoid signaling as a potential target for epigenetic therapy.

Sumoylation is an essential posttranslational modification in eukaryotes that has emerged as an important pathway in oncogenic processes. Most human cancers display hyperactivated sumoylation and many cancer cells are remarkably sensitive to its inhibition, thus supporting application of chemical sumoylation inhibitors in cancer treatment. Here we show, first, that transformed embryonic fibroblasts derived from mice haploinsufficient for Ubc9, the essential and unique gene encoding the SUMO E2 conjugating enzyme, exhibit enhanced proliferation and transformed phenotypes in vitro and as xenografts ex vivo. To then evaluate the possible impact of loss of one Ubc9 allele in vivo, we used a mouse model of intestinal tumorigenesis. We crossed Ubc9+/− mice with mice harboring a conditional ablation of Apc either all along the crypt–villus axis or only in Lgr5+ crypt-based columnar (CBC) cells, the cell compartment that includes the intestinal stem cells proposed as cells-of-origin of intestinal cancer. While Ubc9+/− mice display no overt phenotypes and no globally visible hyposumoylation in cells of the small intestine, we found, strikingly, that, upon loss of Apc in both models, Ubc9+/− mice develop more (>2-fold) intestinal adenomas and show significantly shortened survival. This is accompanied by reduced global sumoylation levels in the polyps, indicating that Ubc9 levels become critical upon oncogenic stress. Moreover, we found that, in normal conditions, Ubc9+/− mice show a moderate but robust (15%) increase in the number of Lgr5+ CBC cells when compared to their wild-type littermates, and further, that these cells display higher degree of stemness and cancer-related and inflammatory gene expression signatures that, altogether, may contribute to enhanced intestinal tumorigenesis. The phenotypes of Ubc9 haploinsufficiency discovered here indicate an unanticipated tumor-suppressive role of sumoylation, one that may have important implications for optimal use of sumoylation inhibitors in the clinic.

The tumor suppressor protein p53 orchestrates cellular responses to a vast number of stresses, with DNA damage and oncogenic activation being some of the best described. The capacity of p53 to control cellular events such as cell cycle progression, DNA repair, and apoptosis, to mention some, has been mostly linked to its role as a transcription factor. However, how p53 integrates different signaling cascades to promote a particular pathway remains an open question. One way to broaden its capacity to respond to different stimuli is by the expression of isoforms that can modulate the activities of the full-length protein. One of these isoforms is p47 (p53/47, Δ40p53, p53ΔN40), an alternative translation initiation variant whose expression is specifically induced by the PERK kinase during the Unfolded Protein Response (UPR) following Endoplasmic Reticulum stress. Despite the increasing knowledge on the p53 pathway, its activity when the translation machinery is globally suppressed during the UPR remains poorly understood. Here, we focus on the expression of p47 and we propose that the alternative initiation of p53 mRNA translation offers a unique condition-dependent mechanism to differentiate p53 activity to control cell homeostasis during the UPR. We also discuss how the manipulation of these processes may influence cancer cell physiology in light of therapeutic approaches.

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.

Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a non-covalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives.

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.

Physiological and pathological conditions that affect the folding capacity of the endoplasmic reticulum (ER) provoke ER stress and trigger the unfolded protein response (UPR). The UPR aims to either restore the balance between newly synthesized and misfolded proteins or if the damage is severe, to trigger cell death. However, the molecular events underlying the switch between repair and cell death are not well understood. The ER-resident chaperone BiP governs the UPR by sensing misfolded proteins and thereby releasing and activating the three mediators of the UPR: PERK, IRE1 and ATF6. PERK promotes G2 cell cycle arrest and cellular repair by inducing the alternative translated p53 isoform p53ΔN40 (p53/47), which activates 14-3-3σ via suppression of p21CDKN1A. Here we show that prolonged ER stress promotes apoptosis via a p53-dependent inhibition of BiP expression. This leads to the release of the pro-apoptotic BH3-only BIK from BiP and activation of apoptosis. Suppression of bip mRNA translation is mediated via the specific binding of p53 to the first 346-nt of the bip mRNA and via a p53 trans-suppression domain located within the first seven N-terminal amino acids of p53ΔN40. This work shows how p53 targets BiP to promote apoptosis during severe ER stress and further illustrates how regulation of mRNA translation has a key role in p53-mediated regulation of gene expression during the UPR.

La enfermedad tromboembolica venosa (ETEV) es una patologia con morbilidad y mortalidad elevadas. Su manejo diagnostico y terapeutico esta en permanente revision. La ausencia de estudios epidemiologicos en nuestro medio implica un desconocimiento de la forma de presentacion, etiologia y recurrencia de la misma. Se propone un estudio descriptivo sobre la poblacion de pacientes con ETEV en seguimiento en la policlinica de Trombosis y Hemostasis del Hospital Pasteur de Montevideo, durante el periodo 2015-2016. Se registraron 35 pacientes con ETEV. La ETEV provocada se asocio predominantemente a factores de riesgo mayor y en los cuales fallo la adecuada prescripcion de tromboprofilaxis. En los pacientes con ETEV no provocada, las trombofilias diagnosticadas fueron el sindrome de anticuerpos anti fosfolipidos, deficit de Antitrombina y mutacion del gen de la protrombina heterocigoto. Los pacientes con ETEV no provocada tuvieron un elevado porcentaje de recurrencias, la mitad de ellas asociadas a trombofilia y la otra mitad sin factor de riesgo predisponente (ETEV idopatica).

Introduccion: La enfermedad tromboembolica venosa (ETEV) constituye la principal causa de muerte prevenible en pacientes hospitalizados. Actualmente se encuentra validado el score de Padua para detectar los pacientes con patologia medica con alto riesgo de ETEV. Objetivos: analizar la prescripcion de tromboprofilaxis farmacologica en pacientes internados por patologia medica y analizar el impacto que genera la realizacion de distintas estrategias para estimular la prescripcion de la misma. Material y metodos: se realizo un corte transversal de todos los pacientes internados en cuidados moderados de medicina en diciembre 2014 aplicando el score de Padua. Tras varias intervenciones de educacion, se realizo un segundo corte transversal un ano despues aplicando la misma herramienta. Resultados: 48/67 pacientes analizados en el primer corte tenian alto riesgo de trombosis, solo 29 tenian indicacion de tromboprofilaxis farmacologica. 32/52 pacientes analizados en el segundo corte tenian indicacion, estando prescripta en 26 de los mismos (p: 0,0062) Discusion: la inclusion del score de Padua en las historias clinicas, la realizacion de instancias de revision de guias, promocion de tromboprofilaxis y concientizacion de la ETEV en el personal medico aumentaron significativamente su prescripcion.

Des fluctuations physiologiques lors de la production et du repliement des proteines, ainsi que des processus pathologiques comme l’infection virale, le vieillissement et les cancers peuvent conduire a un stress du Reticulum Endoplasmique (RE). Il s’agit d’un etat caracterise par l’accumulation de proteines non/mal repliees dans la lumiere du RE, qui declenche la reponse aux proteines depliees, dite UPR (Unfolded Protein Response). Pour retablir l’equilibre proteique, la reponse UPR va inhiber la synthese proteique globale cap-dependante et favoriser la production de proteases et de chaperonnes associees au RE, notamment la proteine BiP qui joue egalement le role de senseur principal de l’UPR. Notre groupe a precedemment montre que lors d’un stress du RE, une isoforme particuliere du suppresseur de tumeur p53, l’isoforme p53ΔN40 (egalement connue sous le nom de p53/47, Δ40p53, ΔNp53 ou p47) est induite selectivement par PERK pour conduire a l’arret de la division cellulaire en G2 et que ceci depend de la suppression de l’expression de p21CDKN1A par l’isoforme longue de p53 (p53FL) avec p53ΔN40 agissant notamment au niveau transcriptionnel et traductionnel.Le sujet principal de mon travail a ete de comprendre comment p53 induit l’apoptose en cas de stress prolonge du RE. J’ai pu montrer que ceci depend de la diminution de l'expression de BiP, via une interaction directe de la proteine p53 avec une petite region de la sequence codante de l'ARNm de BIP. Cette trans-suppression de BiP est mediee par un domaine de 7 acides amines present dans p53FL et aussi dans p53ΔN40. Cette inhibition de l'expression de BiP pendant les tress du RE conduit a une augmentation de l'apoptose par l'activation de la proteine BIK ainsi liberee d’une interaction repressive avec BiP. De plus, BIK est egalement activee pendant le stress du RE par p53FL et/ou p53ΔN40 au niveau transcriptionnel. Mes resultats etablissent pour la premiere fois un lien entre la capacite de liaison a l'ARNm de p53 et le controle de la traduction de cet ARNm avec une reponse cellulaire particuliere.Ce travail montre egalement que p53 controle la traduction de deux ARNm supplementaires par ce qui semble etre deux mecanismes d’action differents. Ces deux mecanismes reposent sur des sequences presentes dans l'ARNm, mais different sur la necessite d'une interaction directe avec la proteine p53. Comme montre pour BiP, la capacite de liaison a l'ARNm de p53 bloque la traduction des ARNm de FGF-2 et de p53. Dans cette categorie, nous pouvons maintenant egalement inclure l'ARNm de MDMX. D’un autre cote, la suppression de la traduction de p21CDKN1An'a pas ete associee a une interaction avec p53, ce qui est aussi le cas pour la suppression de l’expression de MDM2. Les implications physiologiques des suppressions d’expression de MDM2et de MDMX sont discutees.Ces resultats montrent que la suppression de la traduction de l'ARNm mediee par p53 joueun role physiologique majeur lors de l'UPR et soutient le role specifique de la p53ΔN40 en reponse a du stress du RE.

Isoforms derived from alternative splicing, mRNA translation initiation or promoter usage extend the functional repertoire of the p53, p63 and p73 genes family and of their regulators MDM2 and MDMX. Here we show cap-independent translation of an N-terminal truncated isoform of hMDMX, hMDMXp60, which is initiated at the 7th AUG codon downstream of the initiation site for full length hMDMXFL at position +384. hMDMXp60 lacks the p53 binding motif but retains the RING domain and interacts with hMDM2 and hMDMXFL. hMDMXp60 shows higher affinity for hMDM2, as compared to hMDMXFL. In vitro data reveal a positive cooperative interaction between hMDMXp60 and hMDM2 and in cellulo data show that low levels of hMDMXp60 promote degradation of hMDM2 whereas higher levels stabilize hMDM2 and prevent hMDM2-mediated degradation of hMDMXFL. These results describe a novel alternatively translated hMDMX isoform that exhibits unique regulatory activity toward hMDM2 autoubiquitination. The data illustrate how the N-terminus of hMDMX regulates its C-terminal RING domain and the hMDM2 activity.

p53 is activated by different stress and damage pathways and regulates cell biological responses including cell cycle arrest, repair pathways, apoptosis and senescence. Following DNA damage, the levels of p53 increase and via binding to target gene promoters, p53 induces expression of multiple genes including p21(CDKN1A) and mdm2. The effects of p53 on gene expression during the DNA damage response are well mimicked by overexpressing p53 under normal conditions. However, stress to the Endoplasmic Reticulum (ER) and the consequent Unfolded Protein Response (UPR) leads to the induction of the p53/47 isoform that lacks the first 40 aa of p53 and to an active suppression of p21(CDKN1A) transcription and mRNA translation. We now show that during ER stress p53 also suppresses MDM2 protein levels via a similar mechanism. These observations not only raise questions about the physiological role of MDM2 during ER stress but it also reveals a new facet of p53 as a repressor toward 2 of its major target genes during the UPR. As suppression of p21(CDKN1A) and MDM2 protein synthesis is mediated via their coding sequences, it raises the possibility that p53 controls mRNA translation via a common mechanism that might play an important role in how p53 regulates gene expression during the UPR, as compared to the transcription-dependent gene regulation taking place during the DNA damage response.

Abstract Background & Aim: Correlations between DNA variation and human phenotypic differences, such as susceptibility to certain diseases, are not well understood. Polymorphisms can contribute to the observed variation in complex human traits, but their relative contributions remain to be determined. Expression differences between alleles of the same gene have been observed, contributing to phenotypic variation between individuals. Studies showed that variations exist in the relative allelic expression levels in certain genes of heterozygote individuals. Polymorphism at codon 72 of TP53 results in either Arginine or Proline, whose functional significance in carcinogenesis is controversial. Studies showed that the Pro72 is less efficient than Arg72 allele in suppressing cell transformation and inducing apoptosis. We have investigated if the expression of these p53 polymorphs is selectively regulated, using mRNA and DNA from colorectal cancer tissues (CRC). Methods: 28 non-related patients treated at ACCamargo Cancer Center in Brazil were evaluated. DNA and RNA were isolated from frozen tumor tissue using Trizol and phenol/chloroform based protocol. TP53 sequences from DNA and RNA were evaluated by Sanger sequencing and quantified using Pyrosequencing. The assay for allele quantification was designed with the software PyroMark Assay Design Software 2.0, featuring algorithms for full quality control. Results: We found 28 p53 codon 72 SNP heterozygotes tumors and 11 (39.3%) of these showed differential expressions, and most of them preferentially expressed the Pro allele. Pyrogram peak heights are proportional to the frequency of an allele in the sample, providing accurate measures of the proportion of the alleles. Previous results revealed that these SNP expression was significantly associated with gender (P= 0.037), recurrence (P= .005), dirty tumor necrosis (P= 0.025), border pattern of tumor growth (P= 0.05), post chemoradiotherapy use (P= 0.002), p53 immunohistochemistry expression (P= 0.041) and TP53 mutation (P= 0.004), suggesting that the expression of the Pro72 allele is associated with worse tumor features. The expression of Arg72 (OR 3.83; CI 1.02-14.35; P= 0.046) and the TNM grouping stage (OR 7.15; CI 1.45-35.29; P= 0.016) were independent predictors for recurrence. This is the first report describing the differential expression of the p53 codon 72 SNP in CRC and revealed that heterozygotes preferentially express the Pro allele, suggesting that the Pro allele is selectively activated in CRC. Conclusions: The expression of the different p53 polymorphs is selectively regulated in Pro72Arg heterozygotes individuals. Thus, the expression status of the p53 polymorphs, rather than the genotypic status, might be an useful indicator tumor aggressiveness. Citation Format: Ligia P. Oliveira, Bianca G. Lisboa, Ignácio Lopez, Erika MM Santos, Dirce M. Carraro, Fernando A. Soares, Benedito M. Rossi, Renata A. Coudry. Differential expression of codon 72 of TP53 in colorectal tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2014-583

TP53 represents a suitable candidate for a colorectal cancer susceptibility locus. The polymorphism in the p53 72nd codon involves a proline to arginine substitution, leading to changes in gene transcription activity, interaction with other proteins and modulation of apoptosis. Studies evaluating the association between this polymorphism and colorectal cancer (CRC) have shown inconsistent results, and none have evaluated the mRNA status of TP53. The aim of the present study was to evaluate the association between this SNP expression at the mRNA level in CRC samples and patient clinicopathological variables and prognosis, p53 protein expression and TP53 mutation. This is the first report to describe the mRNA expression of p53 codon 72 alleles in CRC. We evaluated 101 non-related patients with CRC treated at the A.C. Camargo Cancer Center in Brazil. RNA was isolated from frozen tumor tissues using a trizol-based protocol. The polymorphism was detected using RT-PCR followed by Sanger sequencing. Associations were analyzed using Pearson's Chi-square or Fisher's exact tests, logistic regression and Cox. This polymorphism was significantly associated with clinicopathological variables related to increased tumor aggressiveness. The expression of Arg72 (OR, 3.83; CI 1.02-14.35; P=0.046) and the TNM stage (OR, 7.15; CI 1.45-35.29; P=0.016) were found to be independent predictors for recurrence. These data suggest that the mRNA expression of the Pro72 allele is associated with less favorable tumor features. The allele frequency of the p53 Pro72 was 0.26. The analysis of mRNA is important to determine the specific contribution of the allele expressed. These results suggest that this polymorphism may play a role in CRC.