RESUMEN
The covalent conjugation of ubiquitin family proteins is a widespread post-translational protein modification. In the ubiquitin family, the ATG8 subfamily is exceptional because it is conjugated mainly to phospholipids. However, it remains unknown whether other ubiquitin family proteins are also conjugated to phospholipids. Here, we report that ubiquitin is conjugated to phospholipids, mainly phosphatidylethanolamine (PE), in yeast and mammalian cells. Ubiquitinated PE (Ub-PE) accumulates at endosomes and the vacuole (or lysosomes), and its level increases during starvation. Ub-PE is also found in baculoviruses. In yeast, PE ubiquitination is catalyzed by the canonical ubiquitin system enzymes Uba1 (E1), Ubc4/5 (E2), and Tul1 (E3) and is reversed by Doa4. Liposomes containing Ub-PE recruit the ESCRT components Vps27-Hse1 and Vps23 in vitro. Ubiquitin-like NEDD8 and ISG15 are also conjugated to phospholipids. These findings suggest that the conjugation to membrane phospholipids is not specific to ATG8 but is a general feature of the ubiquitin family.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Liposomas/metabolismo , Mamíferos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolípidos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.
Asunto(s)
Autofagia , Carcinoma Ductal Pancreático/metabolismo , Lisosomas/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Cromatina/genética , Cromatina/metabolismo , Regulación hacia Abajo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metabolismo Energético , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Lisosomas/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Agregado de Proteínas , Unión Proteica , Proteolisis , Interferencia de ARN , Transducción de Señal , Sirtuina 1/genética , Sirtuina 1/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , TransfecciónRESUMEN
Although cell size regulation is crucial for cellular functions in a variety of organisms from bacteria to humans, the underlying mechanisms remain elusive. Here, we identify Rim21, a component of the pH-sensing Rim101 pathway, as a positive regulator of cell size through a flow cytometry-based genome-wide screen of Saccharomyces cerevisiae deletion mutants. We found that mutants defective in the Rim101 pathway were consistently smaller than wildtype cells in the log and stationary phases. We show that the expression of the active form of Rim101 increased the size of wildtype cells. Furthermore, the size of wildtype cells increased in response to external alkalization. Microscopic observation revealed that this cell size increase was associated with changes in both vacuolar and cytoplasmic volume. We also found that these volume changes were dependent on Rim21 and Rim101. In addition, a mutant lacking Vph1, a component of V-ATPase that is transcriptionally regulated by Rim101, was also smaller than wildtype cells, with no increase in size in response to alkalization. We demonstrate that the loss of Vph1 suppressed the Rim101-induced increase in cell size under physiological pH conditions. Taken together, our results suggest that the cell size of budding yeast is regulated by the Rim101-V-ATPase axis under physiological conditions as well as in response to alkaline stresses.
Asunto(s)
Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Concentración de Iones de Hidrógeno , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Eliminación de SecuenciaRESUMEN
It is now well established that tumours undergo changes in cellular metabolism1. As this can reveal tumour cell vulnerabilities and because many tumours exhibit enhanced glucose uptake2, we have been interested in how tumour cells respond to different forms of sugar. Here we report that the monosaccharide mannose causes growth retardation in several tumour types in vitro, and enhances cell death in response to major forms of chemotherapy. We then show that these effects also occur in vivo in mice following the oral administration of mannose, without significantly affecting the weight and health of the animals. Mechanistically, mannose is taken up by the same transporter(s) as glucose3 but accumulates as mannose-6-phosphate in cells, and this impairs the further metabolism of glucose in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and glycan synthesis. As a result, the administration of mannose in combination with conventional chemotherapy affects levels of anti-apoptotic proteins of the Bcl-2 family, leading to sensitization to cell death. Finally we show that susceptibility to mannose is dependent on the levels of phosphomannose isomerase (PMI). Cells with low levels of PMI are sensitive to mannose, whereas cells with high levels are resistant, but can be made sensitive by RNA-interference-mediated depletion of the enzyme. In addition, we use tissue microarrays to show that PMI levels also vary greatly between different patients and different tumour types, indicating that PMI levels could be used as a biomarker to direct the successful administration of mannose. We consider that the administration of mannose could be a simple, safe and selective therapy in the treatment of cancer, and could be applicable to multiple tumour types.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Manosa/metabolismo , Manosa/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Peso Corporal/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Manosa/administración & dosificación , Manosa/uso terapéutico , Manosa-6-Fosfato Isomerasa/deficiencia , Manosa-6-Fosfato Isomerasa/genética , Manosa-6-Fosfato Isomerasa/metabolismo , Manosafosfatos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/clasificación , Neoplasias/patología , Interferencia de ARN , Proteína bcl-X/metabolismoRESUMEN
Protein-lipid conjugation is a widespread modification involved in many biological processes. Various lipids, including fatty acids, isoprenoids, sterols, glycosylphosphatidylinositol, sphingolipids, and phospholipids, are covalently linked with proteins. These modifications direct proteins to intracellular membranes through the hydrophobic nature of lipids. Some of these membrane-binding processes are reversible through delipidation or by reducing the affinity to membranes. Many signaling molecules undergo lipid modification, and their membrane binding is important for proper signal transduction. The conjugation of proteins to lipids also influences the dynamics and function of organellar membranes. Dysregulation of lipidation has been associated with diseases such as neurodegenerative diseases. In this review, we first provide an overview of diverse forms of protein-lipid conjugation and then summarize the catalytic mechanisms, regulation, and roles of these modifications.Key words: lipid, lipidation, membrane, organelle, protein modification.
Asunto(s)
Ácidos Grasos , Proteínas , Ácidos Grasos/metabolismo , Fosfolípidos/metabolismo , Metabolismo de los Lípidos , Esteroles/metabolismo , Membrana Celular/metabolismoRESUMEN
Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.
Asunto(s)
Autofagia , Sistemas CRISPR-Cas , Amidofosforribosiltransferasa/genética , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genéticaRESUMEN
Protein arginine methylation is a common posttranslational modification catalyzed by a family of the protein arginine methyltransferases (PRMTs). We have previously reported that PRMT1 methylates Forkhead box O transcription factors at two arginine residues within an Akt consensus phosphorylation motif (RxRxxS/T), and that this methylation blocks Akt-mediated phosphorylation of the transcription factors. These findings led us to hypothesize that the functional crosstalk between arginine methylation and phosphorylation could be extended to other Akt target proteins as well as Forkhead box O proteins. Here we identify BCL-2 antagonist of cell death (BAD) as an additional substrate for PRMT1 among several Akt target proteins. We show that PRMT1 specifically binds and methylates BAD at Arg-94 and Arg-96, both of which comprise the Akt consensus phosphorylation motif. Consistent with the hypothesis, PRMT1-mediated methylation of these two arginine residues inhibits Akt-mediated phosphorylation of BAD at Ser-99 in vitro and in vivo. We also demonstrate that the complex formation of BAD with 14-3-3 proteins, which occurs subsequent to Akt-mediated phosphorylation, is negatively regulated by PRMT1. Furthermore, PRMT1 knockdown prevents mitochondrial localization of BAD and its binding to the antiapoptotic BCL-X(L) protein. BAD overexpression causes an increase in apoptosis with concomitant activation of caspase-3, whereas PRMT1 knockdown significantly suppresses these apoptotic processes. Taken together, our results add a new dimension to the complexity of posttranslational BAD regulation and provide evidence that arginine methylation within an Akt consensus phosphorylation motif functions as an inhibitory modification against Akt-dependent survival signaling.
Asunto(s)
Arginina/metabolismo , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Represoras/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Apoptosis , Arginina/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Metilación , Fosforilación , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Represoras/genética , Proteína Letal Asociada a bcl/antagonistas & inhibidores , Proteína Letal Asociada a bcl/genéticaRESUMEN
During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.
Asunto(s)
Autofagosomas , Fosfatos de Fosfatidilinositol , Proteínas Qa-SNARE , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Autofagosomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Humanos , Simulación de Dinámica Molecular , Autofagia/fisiologíaRESUMEN
Conjugation of Atg8-family proteins to phosphatidylethanolamine (PE) is important for autophagosome formation. PE conjugation has been thought to be specific to Atg8 among the ubiquitin-family proteins. However, this dogma has not been experimentally verified. Our recent study revealed that ubiquitin is also conjugated to PE on endosomes and the vacuole (or lysosomes). Other ubiquitin-like proteins, such as NEDD8 and ISG15, also covalently bind to phospholipids. We propose that conjugation to phospholipids could be a common feature of the ubiquitin family.
Asunto(s)
Fosfolípidos , Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Ubiquitinas , Ubiquitina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Relacionadas con la AutofagiaRESUMEN
The covalent attachment of ubiquitin is a common regulatory mechanism in various proteins. Although it has long been thought that the substrates of ubiquitination are limited to proteins, recent studies have changed this view: ubiquitin can be conjugated to lipids, sugars, and nucleotides. Ubiquitin is linked to these substrates by the action of different classes of ubiquitin ligases that have distinct catalytic mechanisms. Ubiquitination of non-protein substrates likely serves as a signal for the recruitment of other proteins to bring about specific effects. These discoveries have expanded the concept of ubiquitination and have advanced our insight into the biology and chemistry of this well-established modification process. In this review we describe the molecular mechanisms and roles of non-protein ubiquitination and discuss the current limitations.
RESUMEN
Ubiquitin is covalently conjugated to phospholipids as well as proteins; however, ubiquitinated phospholipids are less abundant than free ubiquitin and ubiquitinated proteins. Here, we describe protocols to purify ubiquitinated phospholipids in budding yeast and human cells based on their hydrophobicity. Ubiquitinated phospholipids are purified by Triton X-114 phase partitioning and affinity purification and verified by phospholipase D treatment. These protocols enable the detection of tagged as well as endogenous mono- and poly-ubiquitinated phospholipids by immunoblotting. For complete details on the use and execution of this protocol, please refer to Sakamaki et al..1.
Asunto(s)
Saccharomycetales , Humanos , Saccharomycetales/metabolismo , Ubiquitina/metabolismo , Proteínas , Immunoblotting , Línea CelularRESUMEN
The forkhead box O transcription factors convert a variety of external stimuli, including growth factors, nutrients, and oxidative stress, into diverse biological responses through modulation of specific gene expression. Forkhead box O regulation is principally achieved by two distinct mechanisms: post-translational modifications and protein-protein interactions. Among several modifications of forkhead box O factors, we focus on reversible acetylation, describing past research and current advances. In the latter part of this review, we also provide an overview of forkhead box O-binding partners that control the transcriptional activity of forkhead box O factors. These two layers of regulation mostly overlap and thereby enable a more precise fine-tuning of forkhead box O functions involved in metabolism, longevity, and tumor suppression. This article is part of a Special Issue entitled: PI3K-AKT-FoxO axis in cancer and aging.
Asunto(s)
Proteínas 14-3-3/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Acetilación , Animales , Proteína Forkhead Box O1 , Humanos , Modelos Biológicos , Fosforilación , Unión Proteica , Transporte de ProteínasRESUMEN
Hepatic gluconeogenesis is important for the maintenance of blood glucose homeostasis under fasting condition. Hepatocyte nuclear factor 4α (HNF4α) and FOXO1 transcription factors have implicated in this process through transcriptional regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), which are rate-limiting enzymes in gluconeogenesis. In this study, we demonstrate that glycogen synthase kinase 3ß (GSK3ß) regulates the expression of gluconeogenic genes through HNF4α and FOXO1. Silencing of GSK3ß leads to reduction in the expression of gluconeogenic genes, including G6Pase, PEPCK, and peroxisome proliferator-activated receptor γ coactivator-1α. We show that GSK3ß directly binds to both HNF4α and FOXO1. Inhibition of GSK3 by SB-216763 abolishes HNF4α-mediated activation of G6Pase promoter. We also found that overexpression of GSK3ß potentiates G6Pase promoter activation by FOXO1 in a manner dependent on its kinase activity. Treatment of SB-216763 diminishes FOXO1-mediated activation of G6Pase promoter. Taken together, these results reveal a previously unrecognized mechanism for the regulation of gluconeogenic gene expression.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Glucosa-6-Fosfatasa/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas de Choque Térmico/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factores de Transcripción/genética , Western Blotting , Células Cultivadas , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Gluconeogénesis , Glucosa-6-Fosfatasa/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Proteínas de Choque Térmico/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Humanos , Luciferasas/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosfoenolpiruvato Carboxiquinasa (ATP) , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/metabolismoRESUMEN
Loss of pancreatic ß cells is the hallmark of type 1 diabetes, for which provision of insulin is the standard of care. While regenerative and stem cell therapies hold the promise of generating single-source or host-matched tissue to obviate immune-mediated complications, these will still require surgical intervention and immunosuppression. Here we report the development of a high-throughput RNAi screening approach to identify upstream pathways that regulate adult human ß cell quiescence and demonstrate in a screen of the GPCRome that silencing G-protein coupled receptor 3 (GPR3) leads to human pancreatic ß cell proliferation. Loss of GPR3 leads to activation of Salt Inducible Kinase 2 (SIK2), which is necessary and sufficient to drive cell cycle entry, increase ß cell mass, and enhance insulin secretion in mice. Taken together, our data show that targeting the GPR3-SIK2 pathway is a potential strategy to stimulate the regeneration of ß cells.
Asunto(s)
Proliferación Celular/genética , Células Secretoras de Insulina/fisiología , Proteínas Serina-Treonina Quinasas/genética , Receptores Acoplados a Proteínas G/genética , Animales , Humanos , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi's ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Terapia Molecular Dirigida , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Crisis Blástica/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda/genética , Ratones , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27(Kip1) gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependent on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27(Kip1) gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27(Kip1) gene expression.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Línea Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteína Forkhead Box O1 , Humanos , Fosforilación , Poli(ADP-Ribosa) Polimerasa-1 , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transcripción GenéticaRESUMEN
Autophagy is a highly regulated process, and its deregulation can contribute to various diseases, including cancer, immune diseases, and neurodegenerative disorders. Here we describe the design, protocol, and analysis of an imaging-based high-throughput screen with an endogenous autophagy readout. The screen uses a genome-wide siRNA library to identify autophagy regulators in mammalian cells.
Asunto(s)
Autofagia/genética , Técnicas de Silenciamiento del Gen/métodos , ARN Interferente Pequeño/metabolismo , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular , Técnicas de Silenciamiento del Gen/instrumentación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteínas Asociadas a Microtúbulos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Transfección/instrumentación , Transfección/métodosRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is driven by metabolic changes in pancreatic cells caused by oncogenic mutations and dysregulation of p53. PDAC cell lines and PDAC-derived xenografts grow as a result of altered metabolic pathways, changes in stroma, and autophagy. Selective targeting and inhibition of one of these may open avenues for the development of new therapeutic strategies. In this study, we performed a genome-wide siRNA screen in a PDAC cell line using endogenous autophagy as a readout and identified several regulators of autophagy that were required for autophagy-dependent PDAC cell survival. Validation of two promising candidates, MPP7 (MAGUK p55 subfamily member 7, a scaffolding protein involved in cell-cell contacts) and MDH1 (cytosolic Malate dehydrogenase 1), revealed their role in early stages of autophagy during autophagosome formation. MPP7 was involved in the activation of YAP1 (a transcriptional coactivator in the Hippo pathway), which in turn promoted autophagy, whereas MDH1 was required for maintenance of the levels of the essential autophagy initiator serine-threonine kinase ULK1, and increased in the activity upon induction of autophagy. Our results provide a possible explanation for how autophagy is regulated by MPP7 and MDH1, which adds to our understanding of autophagy regulation in PDAC. SIGNIFICANCE: This study identifies and characterizes MPP7 and MDH1 as novel regulators of autophagy, which is thought to be responsible for pancreatic cancer cell survival.
Asunto(s)
Autofagia , Carcinoma Ductal Pancreático/patología , Regulación Neoplásica de la Expresión Génica , Malato Deshidrogenasa/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proliferación Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Malato Deshidrogenasa/antagonistas & inhibidores , Malato Deshidrogenasa/genética , Proteínas de la Membrana/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteínas Señalizadoras YAPRESUMEN
Autophagy is an essential cellular process that degrades cytoplasmic organelles and components. Precise control of autophagic activity is achieved by context-dependent signaling pathways. Recent studies have highlighted the involvement of transcriptional programs during autophagic responses to various signals. Here, we summarize the current understanding of the transcriptional regulation of autophagy.
Asunto(s)
Autofagia , Transcripción Genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas de Ciclo Celular , Código de Histonas , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Macroautophagy/autophagy is an intracellular recycling system that delivers cytoplasmic organelles and materials to lysosomes for degradation. This process is operated by autophagy-related (ATG) genes and tightly controlled by stress-responsive signaling pathways. Our recent study revealed that autophagy programs are transcriptionally suppressed by the BET family protein BRD4. This repression is alleviated during nutrient deprivation through the AMPK-SIRT1 pathway. Our findings therefore provide new insights into the regulation of autophagy.