RESUMEN

The epitranscriptomic writer Alkylation Repair Homolog 8 (ALKBH8) is a transfer RNA (tRNA) methyltransferase that modifies the wobble uridine of selenocysteine tRNA to promote the specialized translation of selenoproteins. Using Alkbh8 deficient (Alkbh8def) mice, we have investigated the importance of epitranscriptomic systems in the response to naphthalene, an abundant polycyclic aromatic hydrocarbon and environmental toxicant. We performed basal lung analysis and naphthalene exposure studies using wild type (WT), Alkbh8def and Cyp2abfgs-null mice, the latter of which lack the cytochrome P450 enzymes required for naphthalene bioactivation. Under basal conditions, lungs from Alkbh8def mice have increased markers of oxidative stress and decreased thioredoxin reductase protein levels, and have reprogrammed gene expression to differentially regulate stress response transcripts. Alkbh8def mice are more sensitive to naphthalene induced death than WT, showing higher susceptibility to lung damage at the cellular and molecular levels. Further, WT mice develop a tolerance to naphthalene after 3 days, defined as resistance to a high challenging dose after repeated exposures, which is absent in Alkbh8def mice. We conclude that the epitranscriptomic writer ALKBH8 plays a protective role against naphthalene-induced lung dysfunction and promotes naphthalene tolerance. Our work provides an early example of how epitranscriptomic systems can regulate the response to environmental stress in vivo.

Asunto(s)

Contaminantes Atmosféricos/toxicidad , Homólogo 8 de AlkB ARNt Metiltransferasa/metabolismo , Epigénesis Genética , Pulmón/metabolismo , Naftalenos/toxicidad , Estrés Oxidativo , Transcriptoma , Homólogo 8 de AlkB ARNt Metiltransferasa/genética , Animales , Sistema Enzimático del Citocromo P-450/genética , Resistencia a Medicamentos , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Procesamiento Postranscripcional del ARN , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo

RESUMEN

Chemical modifications that regulate protein expression at the translational level are emerging as vital components of the cellular stress response. Transfer RNAs (tRNAs) are significant targets for methyl-based modifications, which are catalyzed by tRNA methyltransferases (Trms). Here, Saccharomyces cerevisiae served as a model eukaryote system to investigate the role of 2'-O-ribose tRNA methylation in the cell's response to oxidative stress. Using 2'-O-ribose deletion mutants for trms 3, 7, 13, and 44, in acute and chronic exposure settings, we demonstrate a broad cell sensitivity to oxidative stress-inducing toxicants (i.e., hydrogen peroxide, rotenone, and acetic acid). A global analysis of hydrogen peroxide-induced tRNA modifications shows a complex profile of decreased, or undetectable, 2'-O-ribose modification events in 2'-O-ribose trm mutant strains, providing a critical link between this type of modification event and Trm status post-exposure. Based on the pronounced oxidative stress sensitivity observed for trm7 mutants, we used a bioinformatic tool to identify transcripts as candidates for regulation by Trm7-catalyzed modifications (i.e., enriched in UUC codons decoded by tRNAPheGmAA). This screen identified transcripts linked to diverse biological processes that promote cellular recovery after oxidative stress exposure, including DNA repair, chromatin remodeling, and nutrient acquisition (i.e., CRT10, HIR3, HXT2, and GNP1); moreover, these mutants were also oxidative stress-sensitive. Together, these results solidify a role for TRM3, 7, 13, and 44, in the cellular response to oxidative stress, and implicate 2'-O-ribose tRNA modification as an epitranscriptomic strategy for oxidative stress recovery.

Asunto(s)

ARN de Transferencia/metabolismo , Ribosa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Reparación del ADN/genética , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismo

RESUMEN

Transfer RNAs (tRNAs) undergo extensive chemical modification within cells through the activity of tRNA methyltransferase enzymes (TRMs). Although tRNA modifications are dynamic, how they impact cell behavior after stress and during tumorigenesis is not well understood. This review discusses how tRNA modifications influence the translation of codon-biased transcripts involved in responses to oxidative stress. We further discuss emerging mechanistic details about how aberrant TRM activity in cancer cells can direct programs of codon-biased translation that drive cancer cell phenotypes. The studies reviewed here predict future preventative therapies aimed at augmenting TRM activity in individuals at risk for cancer due to exposure. They further predict that attenuating TRM-dependent translation in cancer cells may limit disease progression while leaving noncancerous cells unharmed.

Asunto(s)

Neoplasias/genética , ARN de Transferencia/genética , Animales , Codón , Regulación Neoplásica de la Expresión Génica , Humanos , Metilación , Neoplasias/metabolismo , Estrés Oxidativo , ARN de Transferencia/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismo

RESUMEN

Ovarian cancer remains the most lethal gynecologic malignancy, and is primarily diagnosed at late stage when considerable metastasis has occurred in the peritoneal cavity. At late stage abdominal cavity ascites accumulation provides a tumor-supporting medium in which cancer cells gain access to growth factors and cytokines that promote survival and metastasis. However, little is known about the redox status of ascites, or whether antioxidant enzymes are required to support ovarian cancer survival during transcoelomic metastasis in this medium. Gene expression cluster analysis of antioxidant enzymes identified two distinct populations of high-grade serous adenocarcinomas (HGSA), the most common ovarian cancer subtype, which specifically separated into clusters based on glutathione peroxidase 3 (GPx3) expression. High GPx3 expression was associated with poorer overall patient survival and increased tumor stage. GPx3 is an extracellular glutathione peroxidase with reported dichotomous roles in cancer. To further examine a potential pro-tumorigenic role of GPx3 in HGSA, stable OVCAR3 GPx3 knock-down cell lines were generated using lentiviral shRNA constructs. Decreased GPx3 expression inhibited clonogenicity and anchorage-independent cell survival. Moreover, GPx3 was necessary for protecting cells from exogenous oxidant insult, as demonstrated by treatment with high dose ascorbate. This cytoprotective effect was shown to be due to GPx3-dependent removal of extracellular H2O2. Importantly, GPx3 was necessary for clonogenic survival when cells were cultured in patient-derived ascites fluid. While oxidation reduction potential (ORP) of malignant ascites was heterogeneous in our patient cohort, and correlated positively with ascites iron content, GPx3 was required for optimal survival regardless of ORP or iron content. Collectively, our data suggest that HGSA ovarian cancers cluster into distinct groups of high and low GPx3 expression. GPx3 is necessary for HGSA ovarian cancer cellular survival in the ascites tumor environment and protects against extracellular sources of oxidative stress, implicating GPx3 as an important adaptation for transcoelomic metastasis.

Asunto(s)

Progresión de la Enfermedad , Espacio Extracelular/metabolismo , Glutatión Peroxidasa/metabolismo , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Antioxidantes/metabolismo , Ascitis/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Células Clonales , Femenino , Humanos , Peróxido de Hidrógeno/toxicidad , Estadificación de Neoplasias , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos

RESUMEN

The translation of mRNA in all forms of life uses a three-nucleotide codon and aminoacyl-tRNAs to synthesize a protein. There are 64 possible codons in the genetic code, with codons for the ∼20 amino acids and 3 stop codons having 1- to 6-fold degeneracy. Recent studies have shown that families of stress response transcripts, termed modification tunable transcripts (MoTTs), use distinct codon biases that match specifically modified tRNAs to regulate their translation during a stress. Similarly, translational reprogramming of the UGA stop codon to generate selenoproteins or to perform programmed translational read-through (PTR) that results in a longer protein, requires distinct codon bias (i.e., more than one stop codon) and, in the case of selenoproteins, a specifically modified tRNA. In an effort to identify transcripts that have codon usage patterns that could be subject to translational control mechanisms, we have used existing genome and transcript data to develop the gene-specific Codon UTilization (CUT) tool and database, which details all 1-, 2-, 3-, 4- and 5-codon combinations for all genes or transcripts in yeast (Saccharomyces cerevisiae), mice (Mus musculus) and rats (Rattus norvegicus). Here, we describe the use of the CUT tool and database to characterize significant codon usage patterns in specific genes and groups of genes. In yeast, we demonstrate how the CUT database can be used to identify genes that have runs of specific codons (e.g., AGA, GAA, AAG) linked to translational regulation by tRNA methyltransferase 9 (Trm9). We further demonstrate how groups of genes can be analyzed to find significant dicodon patterns, with the 80 Gcn4-regulated transcripts significantly (P<0.00001) over-represented with the AGA-GAA dicodon. We have also used the CUT database to identify mouse and rat transcripts with internal UGA codons, with the surprising finding of 45 and 120 such transcripts, respectively, which is much larger than expected. The UGA data suggest that there could be many more translationally reprogrammed transcripts than currently reported. CUT thus represents a multi-species codon-counting database that can be used with mRNA-, translation- and proteomics-based results to better understand and model translational control mechanisms.

Asunto(s)

Codón/genética , Biología Computacional/métodos , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Animales , Codón de Terminación/genética , Bases de Datos Genéticas , Genoma/genética , Ratones , Ratas , Saccharomyces cerevisiae/genética

RESUMEN

Environmental and metabolic sources of reactive oxygen species (ROS) can damage DNA, proteins and lipids to promote disease. Regulation of gene expression can prevent this damage and can include increased transcription, translation and post translational modification. Cellular responses to ROS play important roles in disease prevention, with deficiencies linked to cancer, neurodegeneration and ageing. Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8. Using a new gene targeted knockout mouse cell system, we show that Alkbh8-/- embryonic fibroblasts (MEFs) display elevated ROS levels, increased DNA and lipid damage and hallmarks of cellular stress. We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families. We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS. These studies define Alkbh8 and tRNA modifications as central regulators of cellular oxidative stress responses in mammalian systems. In addition they highlight a new animal model for use in environmental and cancer studies and link translational regulation to the prevention of DNA and lipid damage.

Asunto(s)

Daño del ADN/genética , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismo , Selenocisteína/genética , ARNt Metiltransferasas/genética , Homólogo 8 de AlkB ARNt Metiltransferasa , Animales , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glutatión Peroxidasa/genética , Ratones , Ratones Endogámicos C57BL , Neoplasias/genética , Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , ARN de Transferencia/genética , Reductasa de Tiorredoxina-Disulfuro/genética , Uridina/análogos & derivados , Uridina/farmacología

RESUMEN

tRNA (tRNA) is a key molecule used for protein synthesis, with multiple points of stress-induced regulation that can include transcription, transcript processing, localization and ribonucleoside base modification. Enzyme-catalyzed modification of tRNA occurs at a number of base and sugar positions and has the potential to influence specific anticodon-codon interactions and regulate translation. Notably, altered tRNA modification has been linked to mitochondrial diseases and cancer progression. In this review, specific to Eukaryotic systems, we discuss how recent systems-level analyses using a bioanalytical platform have revealed that there is extensive reprogramming of tRNA modifications in response to cellular stress and during cell cycle progression. Combined with genome-wide codon bias analytics and gene expression studies, a model emerges in which stress-induced reprogramming of tRNA drives the translational regulation of critical response proteins whose transcripts display a distinct codon bias. Termed Modification Tunable Transcripts (MoTTs), (1) we define them as (1) transcripts that use specific degenerate codons and codon biases to encode critical stress response proteins, and (2) transcripts whose translation is influenced by changes in wobble base tRNA modification. In this review we note that the MoTTs translational model is also applicable to the process of stop-codon recoding for selenocysteine incorporation, as stop-codon recoding involves a selective codon bias and modified tRNA to decode selenocysteine during the translation of a key subset of oxidative stress response proteins. Further, we discuss how in addition to RNA modification analytics, the comprehensive characterization of translational regulation of specific transcripts requires a variety of tools, including high coverage codon-reporters, ribosome profiling and linked genomic and proteomic approaches. Together these tools will yield important new insights into the role of translational elongation in cell stress response.

Asunto(s)

Fenómenos Fisiológicos Celulares , Codón/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia/metabolismo , Estrés Fisiológico , Aminoacilación , Animales , Humanos , Neoplasias/metabolismo , ARN de Transferencia/química , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Nucleotide balance is critically important not only in replicating cells but also in quiescent cells. This is especially true in the nervous system, where there is a high demand for adenosine triphosphate (ATP) produced from mitochondria. Mitochondria are particularly prone to oxidative stress-associated DNA damage because nucleotide imbalance can lead to mitochondrial depletion due to low replication fidelity. Failure to maintain nucleotide balance due to genetic defects can result in infantile death; however there is great variability in clinical presentation for particular diseases. This review compares genetic diseases that result from defects in specific nucleotide salvage enzymes and a signaling kinase that activates nucleotide salvage after DNA damage exposure. These diseases include Lesch-Nyhan syndrome, mitochondrial depletion syndromes, and ataxia telangiectasia. Although treatment options are available to palliate symptoms of these diseases, there is no cure. The conclusions drawn from this review include the critical role of guanine nucleotides in preventing neurodegeneration, the limitations of animals as disease models, and the need to further understand nucleotide imbalances in treatment regimens. Such knowledge will hopefully guide future studies into clinical therapies for genetic diseases.

Asunto(s)

Daño del ADN , Enfermedades Neurodegenerativas/metabolismo , Nucleótidos/genética , Adenosina Trifosfato/metabolismo , Animales , Ataxia Telangiectasia/genética , Reparación del ADN , Replicación del ADN , ADN Mitocondrial/genética , Enfermedades Genéticas Congénitas/genética , Humanos , Síndrome de Lesch-Nyhan/genética , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/genética , Estrés Oxidativo , Purinas/metabolismo , Pirimidinas/metabolismo , Transducción de Señal

RESUMEN

Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9-like (hTRM9L/KIAA1456) mRNA is down-regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re-expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence-like G0/G1-arrest and up-regulation of the RB interacting protein LIN9. Additionally, SW620 cells re-expressing hTRM9L did not respond to hypoxia via HIF1-α-dependent induction of GLUT1. Importantly, hTRM9L-negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L-expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1-α-dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L-deficient tumours.

Asunto(s)

Neoplasias del Colon/terapia , Terapia Genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , ARNt Metiltransferasas/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Hipoxia de la Célula , Proliferación Celular , Embrión de Pollo , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Regulación hacia Abajo , Epigénesis Genética , Puntos de Control de la Fase G1 del Ciclo Celular , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Células HCT116 , Células HT29 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Desnudos , Mutación , Proteínas Nucleares/genética , Paromomicina/farmacología , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Transfección , Carga Tumoral , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de Xenoinjerto , ARNt Metiltransferasas/genética

RESUMEN

TNFα is a pleiotropic cytokine that signals for both survival and apoptotic cell fates. It is still unclear that the dual role of TNFα can be regulated in cancer cells. We previously described an apoptotic pathway involving p53→CDIP→TNFα that was activated in response to genotoxic stress. This pathway operated in the presence of JNK activation; therefore, we postulated that CDIP itself could sensitize cells to a TNFα apoptotic cell fate, survival, or death. We show that CDIP mediates sensitivity to TNFα-induced apoptosis and that cancer cells with endogenous CDIP expression are inherently sensitive to the growth-suppressive effects of TNFα in vitro and in vivo. Thus, CDIP expression correlates with sensitivity of cancer cells with TNFα, and CDIP seems to be a regulator of the p53-mediated death versus survival response of cells to TNFα. This CDIP-mediated sensitivity to TNFα-induced apoptosis favors pro- over antiapoptotic program in cancer cells, and CDIP may serve as a predictive biomarker for such sensitivity.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/biosíntesis , Apoptosis/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-8/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , FN-kappa B/biosíntesis , FN-kappa B/genética , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/genética

RESUMEN

The p53 protein is activated by stress signals and exhibits both protective and death-promoting functions that are considered important for its tumor suppressor function. Emerging evidence points toward an additional role for p53 in metabolism. Here, we identify Lpin1 as a p53-responsive gene that is induced in response to DNA damage and glucose deprivation. Lpin1 is essential for adipocyte development and fat metabolism, and mutation in this gene is responsible for the lypodystrophy phenotype in fld mice. We show that p53 and Lpin1 regulate fatty acid oxidation in mouse C2C12 myoblasts. p53 phosphorylation on Ser18 in response to low glucose is ROS and ATM dependent. Lpin1 expression in response to nutritional stress is controlled through the ROS-ATM-p53 pathway and is conserved in human cells. Lpin1 provides a critical link between p53 and metabolism that may be an important component in mediating the tumor suppressor function of p53.

Asunto(s)

Metabolismo Energético , Ácidos Grasos/metabolismo , Glucosa/deficiencia , Mioblastos/metabolismo , Proteínas Nucleares/metabolismo , Estado Nutricional , Estrés Oxidativo , Fosfatidato Fosfatasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Oxidación-Reducción , Fosfatidato Fosfatasa/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Serina , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

Cellular responses to DNA damage can prevent mutations and death. In this study, we have used high throughput screens and developed a comparative genomic approach, termed Functionome mapping, to discover conserved responses to UVC-damage. Functionome mapping uses gene ontology (GO) information to link proteins with similar biological functions from different organisms, and we have used it to compare 303, 311 and 288 UVC-toxicity modulating proteins from Escherichia coli, Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. We have demonstrated that all three organisms use DNA repair, translation and aerobic respiration associated processes to modulate the toxicity of UVC, with these last two categories highlighting the importance of ribosomal proteins and electron transport machinery. Our study has demonstrated that comparative genomic approaches can be used to identify conserved responses to damage, and suggest roles for translational machinery and components of energy metabolism in optimizing the DNA damage response.

Asunto(s)

Respiración de la Célula/genética , Daño del ADN/genética , Reparación del ADN/genética , Biosíntesis de Proteínas/genética , Proteínas/genética , Tolerancia a Radiación/genética , Rayos Ultravioleta , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Genómica/métodos , Ensayos Analíticos de Alto Rendimiento , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de la radiación , Schizosaccharomyces/genética , Schizosaccharomyces/efectos de la radiación , Eliminación de Secuencia

RESUMEN

The small heat shock protein alphaB-crystallin is a molecular chaperone that is induced by stress and protects cells by inhibiting protein aggregation and apoptosis. To identify novel transcriptional regulators of the alphaB-crystallin gene, we examined the alphaB-crystallin promoter for conserved transcription factor DNA-binding elements and identified a putative response element for the p53 tumor suppressor protein. Ectopic expression of wild-type p53 induced alphaB-crystallin mRNA and protein with delayed kinetics compared to p21. Additionally, the induction of alphaB-crystallin by genotoxic stress was inhibited by siRNAs targeting p53. Although the p53-dependent transactivation of an alphaB-crystallin promoter luciferase reporter required the putative p53RE, chromatin immunoprecipitation failed to detect p53 binding to the alphaB-crystallin promoter. These results suggested an indirect mechanism of transactivation involving p53 family members p63 or p73. DeltaNp73 was dramatically induced by p53 in a TAp73-dependent manner, and silencing p73 suppressed the transcriptional activation of alphaB-crystallin by p53. Moreover, ectopic expression of DeltaNp73alpha (but not other p73 isoforms) increased alphaB-crystallin mRNA levels in the absence of p53. Collectively, our results link the molecular chaperone alphaB-crystallin to the cellular genotoxic stress response via a novel mechanism of transcriptional regulation by p53 and p73.

Asunto(s)

Daño del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Nucleares/fisiología , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de Tumor/fisiología , Cadena B de alfa-Cristalina/genética , Sitios de Unión , Carcinoma de Células Transicionales/patología , Línea Celular Tumoral/metabolismo , Doxorrubicina/toxicidad , Genes Reporteros , Proteínas de Choque Térmico HSP27/biosíntesis , Proteínas de Choque Térmico HSP27/genética , Humanos , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/fisiología , Proteínas Recombinantes de Fusión/fisiología , Alineación de Secuencia , Activación Transcripcional , Proteína Tumoral p73 , Neoplasias de la Vejiga Urinaria/patología , Cadena B de alfa-Cristalina/biosíntesis

RESUMEN

The p53 tumor suppressor protein has a well-established role in cell-fate decision-making processes. However, recent discoveries indicate that p53 has a non-tumor-suppressive role. Here we identify guanidinoacetate methyltransferase (GAMT), an enzyme involved in creatine synthesis, as a p53 target gene and a key downstream effector of adaptive response to nutrient stress. We show that GAMT is not only involved in p53-dependent apoptosis in response to genotoxic stress but is important for apoptosis induced by glucose deprivation. Additionally, p53-->GAMT upregulates fatty acid oxidation (FAO) induced by glucose starvation, utilizing this pathway as an alternate ATP-generating energy source. These results highlight that p53-dependent regulation of GAMT allows cells to maintain energy levels sufficient to undergo apoptosis or survival under conditions of nutrient stress. The p53-->GAMT pathway represents a new link between cellular stress responses and processes of creatine synthesis and FAO, demonstrating a further role of p53 in cellular metabolism.

Asunto(s)

Apoptosis/fisiología , Guanidinoacetato N-Metiltransferasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Línea Celular Tumoral , Creatina/biosíntesis , Daño del ADN , Etopósido/farmacología , Ácidos Grasos/metabolismo , Rayos gamma , Regulación de la Expresión Génica , Glucosa/farmacología , Guanidinoacetato N-Metiltransferasa/genética , Células HCT116 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Estrés Oxidativo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genética