RESUMO
p53 is the most commonly mutated gene in human cancers. Two fundamental reasons for this are its long protein isoforms protect from cancer, while its shorter C-terminal isoforms can support cancer and metastasis. Previously, we have shown that the Δ160p53 protein isoform enhances survival and the invasive character of cancer cells. Here, we identified a translation initiation site nine codons downstream of codon 160-the known initiation codon for the translation of Δ160p53-that is recognized by the translation machinery. When translation failed to initiate from AUG160 due to mutation, it initiated from AUG169 instead, producing similar levels of a similar protein, Δ169p53, which promoted cell survival as efficiently as Δ160p53 following DNA damage. Interestingly, almost all mammalian species with an orthologue to human AUG160 also possess one for AUG169, while none of the non-mammalian species lacking AUG160 have AUG169, even if that region of the p53 gene is well conserved. In view of our findings, we do not believe that Δ169p53 acts as a different p53 protein isoform; instead, we propose that the double translation initiation site strengthens the translation of these products with a critical role in cell homeostasis. Future studies will help verify if this is a more general mechanism for the expression of essential proteins in mammals.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Códon de Iniciação/genética , Códon , Isoformas de Proteínas/metabolismo , Mutação , Neoplasias/genética , Biossíntese de ProteínasRESUMO
The mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5' untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions.
Assuntos
Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Regiões 5' não Traduzidas , Animais , Hipóxia Celular/genética , Linhagem Celular , Evolução Molecular , Regulação da Expressão Gênica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Hidrazonas/farmacologia , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/farmacologia , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Dobramento de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Tiazóis/farmacologiaRESUMO
Throughout evolution, eukaryotic cells have devised different mechanisms to cope with stressful environments. When eukaryotic cells are exposed to stress stimuli, they activate adaptive pathways that allow them to restore cellular homeostasis. Most types of stress stimuli have been reported to induce a decrease in overall protein synthesis accompanied by induction of alternative mechanisms of mRNA translation initiation. Here, we present well-studied and recent examples of such stress responses and the alternative translation initiation mechanisms they induce, and discuss the consequences of such regulation for cell homeostasis and oncogenic transformation.
Assuntos
Neoplasias , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Estresse Fisiológico , Humanos , Neoplasias/genética , Biossíntese de Proteínas/genética , Processamento de Proteína Pós-Traducional , Proteômica , Estresse Fisiológico/genéticaRESUMO
Wild-type p53 functions as a tumour suppressor while mutant p53 possesses oncogenic potential. Until now it remains unclear how a single mutation can transform p53 into a functionally distinct gene harbouring a new set of original cellular roles. Here we show that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated full-length p53 mRNA. Cells expressing mutant p53 exhibit "gain-of-function" cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Interestingly, Δ160p53-overexpressing cells behave in a similar manner. In contrast, an exogenous or endogenous mutant p53 that fails to express Δ160p53 due to specific mutations or antisense knock-down loses pro-oncogenic potential. Our data support a model in which "gain-of-function" phenotypes induced by p53 mutations depend on the shorter p53 isoforms. As a conserved wild-type isoform, Δ160p53 has evolved during millions of years. We thus provide a rational explanation for the origin of the tumour-promoting functions of p53 mutations.
Assuntos
Carcinogênese/genética , Mutação , Neoplasias/genética , Biossíntese de Proteínas , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Proteínas de Ligação a DNA , Genes p53 , Humanos , Técnicas de Cultura de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
p53 downstream pathways control G1 and G2 cell-cycle arrest, DNA repair, or apoptosis. However, it is still not clear how cells differentiate the cell-biological outcome of p53 activation in response to different types of stresses. The p53/47 isoform lacks the first 39 amino acids of full-length p53 including the Mdm2 binding site and the first trans-activation domain, and tetramers including p53/47 exhibit altered activity and biochemical properties. Here we show that endoplasmic reticulum stress promotes PERK-dependent induction of p53/47 mRNA translation and p53/47 homo-oligomerization. p53/47 induces 14-3-3sigma and G2 arrest but does not affect G1 progression. This is contrary to p53FL, which promotes G1 arrest but has no effect on the G2. These results show a unique role for p53/47 in the p53 pathway and illustrate how a cellular stress leads to a defined cell-biological outcome through expression of a p53 isoform.
Assuntos
Retículo Endoplasmático/metabolismo , Fase G2/fisiologia , Biossíntese de Proteínas , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Estresse Fisiológico , Proteína Supressora de Tumor p53/metabolismo , Proteínas 14-3-3 , Apoptose/fisiologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Exonucleases/genética , Exonucleases/metabolismo , Exorribonucleases , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
The E3 ubiquitin ligase Mdm2 is a focal regulator of p53 tumour suppressor activity. It binds p53, promoting its polyubiquitination and degradation, and also controls p53 synthesis. However, it is not known how this dual function of Mdm2 on p53 synthesis and degradation is achieved. Here we show that the p53 mRNA region encoding the Mdm2-binding site interacts directly with the RING domain of Mdm2. This impairs the E3 ligase activity of Mdm2 and promotes p53 mRNA translation. We also show that introduction of cancer-derived single silent point-mutations in the p53 mRNA weakens its binding to Mdm2 and results in reduced p53 activity. These data are consistent with a mechanism by which changes in silent nucleotides can affect the function of the encoded protein, and indicate that Mdm2-mediated control of p53 synthesis and degradation has evolved in the p53 mRNA sequence and its encoded amino acids.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: There are a growing number of reports on the sub-physiological temperature culturing of mammalian cells for increased recombinant protein yields. However, the effect varies and the reasons for the enhancement are not fully elucidated. Expression of cold-inducible RNA-binding protein (cirp, also called cirbp or hnRNP A18) is known to be induced in response to mild, but not severe, hypothermia in mammalian cells. To clarify the molecular mechanism underlying the induction and to exploit this to improve the productivity of recombinant proteins, we tried to identify the regulatory sequence(s) in the 5' flanking region of the mouse cirp gene. RESULTS: By transiently transfecting HEK293 cells with plasmids expressing chloramphenicol acetyltransferase as a reporter, we found that the cirp 5' flanking region octanucleotide 5'-TCCCCGCC-3' is a mild-cold responsive element (MCRE). When 3 copies of MCRE were placed upstream of the CMV promoter and used in transient transfection, reporter gene expression was increased 3- to 7-fold at 32°C relative to 37°C in various cell lines including HEK293, U-2 OS, NIH/3T3, BALB/3T3 and CHO-K1 cells. In stable transfectants, MCRE also enhanced the reporter gene expression at 32°C, although more copy numbers of MCRE were necessary. Sp1 transcription factor bound to MCRE in vitro. Immunohistochemistry and chromatin immunoprecipitation assays demonstrated that more Sp1, but not Sp3, was localized in the nucleus to bind to the cirp regulatory region containing MCRE at 32°C than 37°C. Overexpression of Sp1 protein increased the expression of endogenous Cirp as well as a reporter gene driven by the 5' flanking region of the cirp gene, and down-regulation of Sp1 had the opposite effect. Mutations within the MCRE sequence in the 5' flanking region abolished the effects of Sp1 on the reporter gene expression both at 37°C and 32°C. CONCLUSIONS: Cold-induced, as well as constitutive, expression of cirp is dependent, at least partly, on MCRE and Sp1. The present novel enhancer permits conditional high-level gene expression at moderately low culture temperatures and could be utilized to increase the yield of recombinant proteins in mammalian cells.
Assuntos
Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição Sp1/metabolismo , Região 5'-Flanqueadora , Animais , Células 3T3 BALB , Células CHO , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Cricetinae , Cricetulus , Regulação para Baixo , Elementos Facilitadores Genéticos , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Camundongos , Mutação , Células NIH 3T3 , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Temperatura , TransfecçãoRESUMO
Ribosome biogenesis is an energetically expensive program that is dictated by nutrient availability. Here we report that nutrient deprivation severely impairs precursor ribosomal RNA (pre-rRNA) processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, pre-rRNAs stored under starvation are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient stress leads to perturbed ribosome assembly upon nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine is dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which is in turn dependent on Raf/MEK/ERK signaling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that tumor cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.
Assuntos
Glutamina , Neoplasias , Glutamina/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Inibidores da Síntese de Ácido Nucleico , Precursores de RNA/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The ATM kinase and p53 are key tumor suppressor factors that control the genotoxic stress response pathway. The ATM substrate Mdm2 controls p53 activity by either targeting p53 for degradation or promoting its synthesis by binding the p53 mRNA. The physiological role and regulation of Mdm2's dual function toward p53 is not known. Here we show that ATM-dependent phosphorylation of Mdm2 at Ser395 is required for the p53 mRNA-Mdm2 interaction. This event also promotes SUMO-conjugation of Mdm2 and its nucleoli accumulation. Interfering with the p53 mRNA-Mdm2 interaction prevents p53 stabilization and activation following DNA damage. These results demonstrate how ATM activity switches Mdm2 from a negative to a positive regulator of p53 via the p53 mRNA.
Assuntos
Dano ao DNA , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Humanos , Fosforilação , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The p53 protein, like any other protein, cannot be made in the cell without RNA. And even once made, the p53 protein will be more rapidly degraded without the p53 RNA. Furthermore, the p53 RNA helps deciding which p53 isoform should be produced and under which cell conditions. Mutant p53 mRNA codes for an unstable and inactive protein. These matters are discussed in this article as well as the recent reports on p53 RNA mutations, interacting-proteins, 3' processing and 5'-3' loop.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regiões não Traduzidas/genética , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Mensageiro/metabolismoRESUMO
SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinesinas/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas Nucleares/genética , RNA Mensageiro/metabolismoRESUMO
The E3 ligase Mdm2 is a key regulator of p53 activity via a complex regulatory feedback system that involves all levels of expression control including transcription, mRNA translation and protein degradation. Best known is the effect of p53 on Mdm2 transcription and the capacity of Mdm2 to target p53 for degradation, but more recently the role of Mdm2 as a positive regulator of p53 activity has also started to emerge. Mdm2 stimulates p53 mRNA translation by binding the p53 mRNA and, interestingly, this interaction also suppresses Mdm2's capacity to promote p53 polyubiquitination and degradation. Another interesting aspect of the p53 mRNA-Mdm2 interaction is that the p53 mRNA sequence encoding the amino acids which bind the N-terminus of Mdm2 is the same that interacts with the Mdm2 RING domain. Indeed, the regulatory elements for controlling Mdm2-dependent expression of p53 are derived from the same p53 genomic sequence. In addition, the RNA binding and the E3 ligase domain of Mdm2 overlap, indicati that the two functions of Mdm2 to control p53 synthesis and degradation have co-evolved in parallel in both p53 and Mdm2. Here we illustrate how the p53-Mdm2 protein-protein and p53 mRNA-Mdm2 interactions affect Mdm2-mediated control of p53 expression using the Phe19Ala p53 mutant. We discuss how the new insights into the regulation of p53 expression levels can help to shed light on the origin of this elegant feedback system and on the function of Mdm2 isoforms.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genética , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Partial degradation or regulated ubiquitin proteasome-dependent processing by the 26 S proteasome has been demonstrated, but the underlying molecular mechanisms and the prevalence of this phenomenon remain obscure. Here we show that the Gly-Ala repeat (GAr) sequence of EBNA1 affects processing of substrates via the ubiquitin-dependent degradation pathway in a substrate- and position-specific fashion. GAr-mediated increase in stability of proteins targeted for degradation via the 26 S proteasome was associated with a fraction of the substrates being partially processed and the release of the free GAr. The GAr did not cause a problem for the proteolytic activity of the proteasome, and its fusion to the N terminus of p53 resulted in an increase in the rate of degradation of the entire chimera. Interestingly the GAr had little effect on the stability of EBNA1 protein itself, and targeting EBNA1 for 26 S proteasome-dependent degradation led to its complete degradation. Taken together, our data suggest a model in which the GAr prevents degradation or promotes endoproteolytic processing of substrates targeted for the 26 S proteasome by interfering with the initiation step of substrate unfolding. These results will help to further understand the underlying mechanisms for partial proteasome-dependent degradation.
Assuntos
Alanina/química , Glicina/química , Complexo de Endopeptidases do Proteassoma/química , Genes p53 , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Conformação Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , Transfecção , Ubiquitina/químicaRESUMO
The platinum(II)-based complex cisplatin is one of the most frequently used antitumour agents; however, a high incidence of harmful side effects and the frequent emergence of acquired resistance are the major clinical problems. The novel platinum(IV)-based complex LA-12 exhibits a high efficacy against cancer cell lines, including cisplatin-insensitive cells, but the mechanisms by which LA-12 perturbs cell growth are unclear. We tested the effects of LA-12 on the p53 response and demonstrate that LA-12 induces unique changes in the profile of gene expression compared with cisplatin and doxorubicin. Furthermore, the ability of LA-12 to disrupt cellular proliferation is greatly enhanced by the expression of p53 and p53/47 indicating both p53-dependent and p53-independent effects of LA-12. Exposure of the human cancer cell lines H1299, A2780, BT549 and BT474 to LA-12 alters the expression of p53 and p53/47 in both a time-dependent and dose-dependent manner. Treatment of cells with a low concentration of the drug results in accumulation of p53 and p53/47 concomitant with their posttranslational modification, whereas a high dose results in the disappearance of both the forms of p53. The distinct p53 activation profile of LA-12 compared with cisplatin and doxorubicin provides a molecular explanation for the ability of this drug to treat cisplatin-resistant cells and indicates its potential usefulness as an alternative antitumour agent in first-line therapy or as a second-line therapy in patients with acquired cisplatin resistance.