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1.
Cancer Res ; 80(21): 4693-4706, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32963011

RESUMO

Multiple myeloma is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins, which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of multiple myeloma is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of multiple myeloma cells. One of these is FAM46C that is lost in more than 10% of patients with multiple myeloma. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A, which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitution of FAM46C in multiple myeloma cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased translation of IgG mRNA, but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER bound protein FNDC3A to reside in the cytoplasmic side of the ER. FNDC3A was lost in some multiple myeloma cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that multiple myeloma cells remodel their trafficking machinery to cope with ER stress. SIGNIFICANCE: This study identifies a new multiple myeloma-specific tumor suppressor complex that regulates autophagy and unconventional secretion, highlighting the sensitivity of multiple myeloma cells to the accumulation of protein aggregates.


Assuntos
Fibronectinas/metabolismo , Mieloma Múltiplo/patologia , Nucleotidiltransferases/metabolismo , Agregação Patológica de Proteínas/metabolismo , Animais , Autofagia/fisiologia , Genes Supressores de Tumor , Xenoenxertos , Humanos , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Nucleotidiltransferases/genética , Agregados Proteicos/fisiologia , Transporte Proteico/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
2.
Dev Comp Immunol ; 109: 103697, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32330465

RESUMO

The transition from a naïve to an effector T cell is an essential event that requires metabolic reprogramming. We have recently demonstrated that the rapid metabolic changes that occur following stimulation of naïve T cells require the translation of preexisting mRNAs. Here, we provide evidence that translation regulates the metabolic asset of effector T cells. By performing ribosome profiling in human CD4+ Th1 cells, we show that the metabolism of glucose, fatty acids and pentose phosphates is regulated at the translational level. In Th1 cells, each pathway has at least one enzyme regulated at the translational level and selected enzymes have high translational efficiencies. mRNA expression does not predict protein expression. For instance, PKM2 mRNA is equally present in naïve T and Th1 cells, but the protein is abundant only in Th1. 5'-untranslated regions (UTRs) may partly account for this regulation. Overall we suggest that immunometabolism is controlled by translation.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Perfilação da Expressão Gênica/métodos , Redes e Vias Metabólicas/genética , Biossíntese de Proteínas/genética , Ribossomos/genética , Células Th1/metabolismo , Regiões 5' não Traduzidas/genética , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/genética , Células Cultivadas , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq/métodos , Ribossomos/metabolismo , Células Th1/citologia
3.
Elife ; 92020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32207685

RESUMO

Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles since MECP2 is expressed throughout the brain and its duplication leads to severe neurological conditions as well. Herein, we use the AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic Mecp2 mutant mice significantly improved locomotor activity, lifespan and gene expression normalization. Remarkably, PHP.eB-iMecp2 administration was well tolerated in female Mecp2 mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male Mecp2 mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of iMecp2 provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain.


Assuntos
Encéfalo/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Mutação/genética , Síndrome de Rett/genética , Animais , Modelos Animais de Doenças , Expressão Gênica/fisiologia , Regulação da Expressão Gênica , Terapia Genética/métodos , Camundongos Transgênicos , Transgenes/genética
4.
Cells ; 9(1)2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936702

RESUMO

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
5.
Dev Biol ; 455(1): 100-111, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31283922

RESUMO

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster model of eIF6 upregulation, leading to a boost in general translation and the shut-down of the ecdysone biosynthetic pathway. Indeed, translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed Programmed Cell Death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ecdisona/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas/genética , Animais , Animais Geneticamente Modificados , Apoptose/genética , Linhagem Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Front Genet ; 9: 533, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30498507

RESUMO

Ribosomes have been long considered as executors of the translational program. The fact that ribosomes can control the translation of specific mRNAs or entire cellular programs is often neglected. Ribosomopathies, inherited diseases with mutations in ribosomal factors, show tissue specific defects and cancer predisposition. Studies of ribosomopathies have paved the way to the concept that ribosomes may control translation of specific mRNAs. Studies in Drosophila and mice support the existence of heterogeneous ribosomes that differentially translate mRNAs to coordinate cellular programs. Recent studies have now shown that ribosomal activity is not only a critical regulator of growth but also of metabolism. For instance, glycolysis and mitochondrial function have been found to be affected by ribosomal availability. Also, ATP levels drop in models of ribosomopathies. We discuss findings highlighting the relevance of ribosome heterogeneity in physiological and pathological conditions, as well as the possibility that in rate-limiting situations, ribosomes may favor some translational programs. We discuss the effects of ribosome heterogeneity on cellular metabolism, tumorigenesis and aging. We speculate a scenario in which ribosomes are not only executors of a metabolic program but act as modulators.

9.
Mol Cell Biol ; 38(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30201806

RESUMO

The translational capability of ribosomes deprived of specific nonfundamental ribosomal proteins may be altered. Physiological mechanisms are scanty, and it is unclear whether free ribosomal proteins can cross talk with the signaling machinery. RACK1 (receptor for activated C kinase 1) is a highly conserved scaffold protein, located on the 40S subunit near the mRNA exit channel. RACK1 is involved in a variety of intracellular contexts, both on and off the ribosomes, acting as a receptor for proteins in signaling, such as the protein kinase C (PKC) family. Here we show that the binding of RACK1 to ribosomes is essential for full translation of capped mRNAs and efficient recruitment of eukaryotic initiation factor 4E (eIF4E). In vitro, when RACK1 is partially depleted, supplementing the ribosome machinery with wild-type RACK1 restores the translational capability, whereas the addition of a RACK1 mutant that is unable to bind ribosomes does not. Outside the ribosome, RACK1 has a reduced half-life. By accumulating in living cells, free RACK1 exerts an inhibitory phenotype, impairing cell cycle progression and repressing global translation. Here we present RACK1 binding to ribosomes as a crucial way to regulate translation, possibly through interaction with known partners on or off the ribosome that are involved in signaling.

10.
Cell Metab ; 28(6): 895-906.e5, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30197303

RESUMO

Naive T cells respond to T cell receptor (TCR) activation by leaving quiescence, remodeling metabolism, initiating expansion, and differentiating toward effector T cells. The molecular mechanisms coordinating the naive to effector transition are central to the functioning of the immune system, but remain elusive. Here, we discover that T cells fulfill this transitional process through translational control. Naive cells accumulate untranslated mRNAs encoding for glycolysis and fatty acid synthesis factors and possess a translational machinery poised for immediate protein synthesis. Upon TCR engagement, activation of the translational machinery leads to synthesis of GLUT1 protein to drive glucose entry. Subsequently, translation of ACC1 mRNA completes metabolic reprogramming toward an effector phenotype. Notably, inhibition of the eIF4F complex abrogates lymphocyte metabolic activation and differentiation, suggesting ACC1 to be a key regulatory node. Thus, our results demonstrate that translation is a direct mediator of T cell metabolism and indicate translation factors as targets for novel immunotherapeutic approaches.


Assuntos
Acetil-CoA Carboxilase/biossíntese , Linfócitos T CD4-Positivos/metabolismo , Ácidos Graxos/metabolismo , Transportador de Glucose Tipo 1/biossíntese , Glicólise , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Proliferação de Células , Fator de Iniciação 4F em Eucariotos/antagonistas & inibidores , Humanos , Ativação Linfocitária , Biossíntese de Proteínas , Subpopulações de Linfócitos T/citologia
11.
PLoS Genet ; 13(1): e1006552, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28056084

RESUMO

Ribosomopathies are a family of inherited disorders caused by mutations in genes necessary for ribosomal function. Shwachman-Diamond Bodian Syndrome (SDS) is an autosomal recessive disease caused, in most patients, by mutations of the SBDS gene. SBDS is a protein required for the maturation of 60S ribosomes. SDS patients present exocrine pancreatic insufficiency, neutropenia, chronic infections, and skeletal abnormalities. Later in life, patients are prone to myelodisplastic syndrome and acute myeloid leukemia (AML). It is unknown why patients develop AML and which cellular alterations are directly due to the loss of the SBDS protein. Here we derived mouse embryonic fibroblast lines from an SbdsR126T/R126T mouse model. After their immortalization, we reconstituted them by adding wild type Sbds. We then performed a comprehensive analysis of cellular functions including colony formation, translational and transcriptional RNA-seq, stress and drug sensitivity. We show that: 1. Mutant Sbds causes a reduction in cellular clonogenic capability and oncogene-induced transformation. 2. Mutant Sbds causes a marked increase in immature 60S subunits, limited impact on mRNA specific initiation of translation, but reduced global protein synthesis capability. 3. Chronic loss of SBDS activity leads to a rewiring of gene expression with reduced ribosomal capability, but increased lysosomal and catabolic activity. 4. Consistently with the gene signature, we found that SBDS loss causes a reduction in ATP and lactate levels, and increased susceptibility to DNA damage. Combining our data, we conclude that a cell-specific fragile phenotype occurs when SBDS protein drops below a threshold level, and propose a new interpretation of the disease.


Assuntos
Homeostase , Fenótipo , Proteínas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Transformação Celular Neoplásica , Dano ao DNA , Fibroblastos/metabolismo , Ácido Láctico/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo
12.
Sci Rep ; 5: 15401, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26486184

RESUMO

Ribosomes function as platforms for binding of other molecules, but technologies for studying this process are lacking. Therefore we developed iRIA (in vitro Ribosomes Interaction Assay). In approach I, Artemia salina ribosomes spotted on solid phase are used for binding picomoles of analytes; in approach II, cellular extracts allow the measurement of ribosome activity in different conditions. We apply the method to analyze several features of eIF6 binding to 60S subunits. By approach I, we show that the off-rate of eIF6 from preribosomes is slower than from mature ribosomes and that its binding to mature 60S occurs in the nM affinity range. By approach II we show that eIF6 binding sites on 60S are increased with mild eIF6 depletion and decreased in cells that are devoid of SBDS, a ribosomal factor necessary for 60S maturation and involved in Swachman Diamond syndrome. We show binding conditions to immobilized ribosomes adaptable to HT and quantify free ribosomes in cell extracts. In conclusion, we suggest that iRIA will greatly facilitate the study of interactions on the ribosomal surface.


Assuntos
Fatores de Iniciação em Eucariotos/genética , Técnicas In Vitro/métodos , Ribossomos/genética , Animais , Artemia/genética , Artemia/metabolismo , Sítios de Ligação , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Ligação Proteica/genética , Ribossomos/metabolismo
13.
Cell Mol Life Sci ; 70(8): 1439-50, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23212600

RESUMO

The receptor for activated C-kinase 1 (RACK1) is a conserved structural protein of 40S ribosomes. Strikingly, deletion of RACK1 in yeast homolog Asc1 is not lethal. Mammalian RACK1 also interacts with many nonribosomal proteins, hinting at several extraribosomal functions. A knockout mouse for RACK1 has not previously been described. We produced the first RACK1 mutant mouse, in which both alleles of RACK1 gene are defective in RACK1 expression (ΔF/ΔF), in a pure C57 Black/6 background. In a sample of 287 pups, we observed no ΔF/ΔF mice (72 expected). Dissection and genotyping of embryos at various stages showed that lethality occurs at gastrulation. Heterozygotes (ΔF/+) have skin pigmentation defects with a white belly spot and hypopigmented tail and paws. ΔF/+ have a transient growth deficit (shown by measuring pup size at P11). The pigmentation deficit is partly reverted by p53 deletion, whereas the lethality is not. ΔF/+ livers have mild accumulation of inactive 80S ribosomal subunits by polysomal profile analysis. In ΔF/+ fibroblasts, protein synthesis response to extracellular and pharmacological stimuli is reduced. These results highlight the role of RACK1 as a ribosomal protein converging signaling to the translational apparatus.


Assuntos
Neuropeptídeos/genética , Pigmentação , Biossíntese de Proteínas , Animais , Células Cultivadas , Perda do Embrião/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/metabolismo , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Quinase C Ativada , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
14.
Autophagy ; 7(8): 907-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21490425

RESUMO

Polyglutamine diseases are accompanied by large-scale alterations of transcription that may be responsible for neurodegeneration. We have monitored early transcriptional alterations in a Drosophila model for dentatorubral-pallidoluysian atrophy (DRPLA) and reported the critical downregulation of the fat tumor suppressor gene. We show that, besides partially mediating neurodegeneration in the Drosophila model for DRPLA, fat and the downstream hippo pathway are essential for adult neuronal homeostasis. This function of the Fat/Hippo pathway is independent of the well-established actions on proliferation and cell polarity; rather it has an impact upon autophagy, which is blocked and ineffective in fat/hippo mutants. These findings reveal the unexpected role in postmitotic neuronal homeostasis of a tumor suppressor pathway and hint at a new player involved directly or indirectly in the regulation of autophagy.


Assuntos
Autofagia , Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Degeneração Neural/patologia , Neurônios/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Envelhecimento/patologia , Animais , Citoproteção , Drosophila melanogaster/citologia , Modelos Biológicos , Degeneração Neural/metabolismo , Neurônios/metabolismo , Proteínas Supressoras de Tumor/metabolismo
15.
EMBO J ; 30(5): 945-58, 2011 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-21278706

RESUMO

Large alterations in transcription accompany neurodegeneration in polyglutamine (polyQ) diseases. These pathologies manifest both general polyQ toxicity and mutant protein-specific effects. In this study, we report that the fat tumour suppressor gene mediates neurodegeneration induced by the polyQ protein Atrophin. We have monitored early transcriptional alterations in a Drosophila model of Dentatorubral-pallidoluysian Atrophy and found that polyQ Atrophins downregulate fat. Fat protects from neurodegeneration and Atrophin toxicity through the Hippo kinase cascade. Fat/Hippo signalling does not provoke neurodegeneration by stimulating overgrowth; rather, it alters the autophagic flux in photoreceptor neurons, thereby affecting cell homeostasis. Our data thus provide a crucial insight into the specific mechanism of a polyQ disease and reveal an unexpected neuroprotective role of the Fat/Hippo pathway.


Assuntos
Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Epilepsias Mioclônicas Progressivas/metabolismo , Epilepsias Mioclônicas Progressivas/patologia , Proteínas do Tecido Nervoso/farmacologia , Peptídeos/genética , Animais , Autofagia , Moléculas de Adesão Celular/genética , Modelos Animais de Doenças , Drosophila/genética , Proteínas de Drosophila/genética , Técnicas Imunoenzimáticas , Epilepsias Mioclônicas Progressivas/genética , Degeneração Neural , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais , Transcrição Genética
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