Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.484
Filtrar
1.
Genes Dev ; 34(5-6): 302-320, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32029452

RESUMO

ADP-ribosylation (ADPRylation) is a posttranslational modification of proteins discovered nearly six decades ago, but many important questions remain regarding its molecular functions and biological roles, as well as the activity of the ADP-ribose (ADPR) transferase enzymes (PARP family members) that catalyze it. Growing evidence indicates that PARP-mediated ADPRylation events are key regulators of the protein biosynthetic pathway, leading from rDNA transcription and ribosome biogenesis to mRNA synthesis, processing, and translation. In this review we describe the role of PARP proteins and ADPRylation in all facets of this pathway. PARP-1 and its enzymatic activity are key regulators of rDNA transcription, which is a critical step in ribosome biogenesis. An emerging role of PARPs in alternative splicing of mRNAs, as well as direct ADPRylation of mRNAs, highlight the role of PARP members in RNA processing. Furthermore, PARP activity, stimulated by cellular stresses, such as viral infections and ER stress, leads to the regulation of mRNA stability and protein synthesis through posttranscriptional mechanisms. Dysregulation of PARP activity in these processes can promote disease states. Collectively, these results highlight the importance of PARP family members and ADPRylation in gene regulation, mRNA processing, and protein abundance. Future studies in these areas will yield new insights into the fundamental mechanisms and a broader utility for PARP-targeted therapeutic agents.


Assuntos
ADP-Ribosilação/fisiologia , Expressão Gênica/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Biossíntese de Proteínas/fisiologia , Proteostase/fisiologia , Animais , Humanos , Processamento de Proteína Pós-Traducional , RNA/metabolismo
2.
Nat Rev Mol Cell Biol ; 21(4): 183-203, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31937935

RESUMO

The mTOR pathway integrates a diverse set of environmental cues, such as growth factor signals and nutritional status, to direct eukaryotic cell growth. Over the past two and a half decades, mapping of the mTOR signalling landscape has revealed that mTOR controls biomass accumulation and metabolism by modulating key cellular processes, including protein synthesis and autophagy. Given the pathway's central role in maintaining cellular and physiological homeostasis, dysregulation of mTOR signalling has been implicated in metabolic disorders, neurodegeneration, cancer and ageing. In this Review, we highlight recent advances in our understanding of the complex regulation of the mTOR pathway and discuss its function in the context of physiology, human disease and pharmacological intervention.


Assuntos
Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Envelhecimento/metabolismo , Animais , Autofagia/fisiologia , Humanos , Doenças Metabólicas/metabolismo , Neoplasias/metabolismo , Estado Nutricional/fisiologia , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia
3.
Nat Neurosci ; 23(2): 281-292, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959934

RESUMO

New protein synthesis is known to be required for the consolidation of memories, yet existing methods of blocking translation lack spatiotemporal precision and cell-type specificity, preventing investigation of cell-specific contributions of protein synthesis. Here we developed a combined knock-in mouse and chemogenetic approach for cell-type-specific drug-inducible protein synthesis inhibition that enables rapid and reversible phosphorylation of eukaryotic initiation factor 2α, leading to inhibition of general translation by 50% in vivo. We use cell-type-specific drug-inducible protein synthesis inhibition to show that targeted protein synthesis inhibition pan-neuronally and in excitatory neurons in the lateral amygdala (LA) impaired long-term memory. This could be recovered with artificial chemogenetic activation of LA neurons, although at the cost of stimulus generalization. Conversely, genetically reducing phosphorylation of eukaryotic initiation factor 2α in excitatory neurons in the LA enhanced memory strength but reduced memory fidelity and behavioral flexibility. Our findings provide evidence for a cell-specific translation program during consolidation of threat memories.


Assuntos
Tonsila do Cerebelo/fisiologia , Consolidação da Memória/fisiologia , Neurônios/fisiologia , Biossíntese de Proteínas/fisiologia , Animais , Fator de Iniciação 2 em Eucariotos/metabolismo , Camundongos
4.
Proc Natl Acad Sci U S A ; 117(1): 761-770, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871206

RESUMO

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga Chlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show that Chlamydomonas VIG1, an ortholog of the Drosophila melanogaster Vasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. A VIG1-deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavage-mediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts.


Assuntos
Chlamydomonas reinhardtii/fisiologia , Proteínas de Plantas/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas Argonauta/metabolismo , Cicloeximida/farmacologia , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas , Íntrons/genética , Mutação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Polirribossomos/genética , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
5.
PLoS Pathog ; 15(12): e1008155, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31856218

RESUMO

Cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs. During viral infections, this includes the expression of antiviral genes that are essential to combat the pathogen. Transcribed mRNAs are bound and escorted to the cytoplasm by the cap-binding complex (CBC). We recently identified a protein complex consisting of NCBP1 and NCBP3 that, under physiological conditions, has redundant function to the canonical CBC, consisting of NCBP1 and NCBP2. Here, we provide evidence that NCBP3 is essential to mount a precise and appropriate antiviral response. Ncbp3-deficient cells allow higher virus growth and elicit a reduced antiviral response, a defect happening on post-transcriptional level. Ncbp3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge. While NCBP3 appeared to be particularly important during viral infections, it may be more broadly involved to ensure proper protein expression.


Assuntos
Infecções por Orthomyxoviridae/imunologia , Proteínas de Ligação ao Cap de RNA/imunologia , Proteínas de Ligação ao Cap de RNA/metabolismo , Animais , Vírus da Influenza A/imunologia , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/metabolismo , Biossíntese de Proteínas/fisiologia
6.
Invest Ophthalmol Vis Sci ; 60(14): 4849-4857, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31747684

RESUMO

Purpose: We reported previously that retinas of mice with inherited retinal degeneration make less protein than retinas of normal mice. Despite recent studies suggesting that diminished protein synthesis rates may contribute to neurologic disorders, a direct link between protein synthesis rates and the progression of neurodegeneration has not been established. Moreover, it remains unclear whether reduced protein synthesis could be involved in retinal pathogenesis. Dysregulation of AKT/mTOR signaling has been reported in the retina during retinal degeneration, but to what extent this signaling contributes to translational attenuation in these mice remains uncertain. Methods: C57BL/6J and rd16 mice were subcutaneously injected with anisomycin to chronically inhibit protein synthesis rates. An AAV2 construct encoding constitutively active 4ebp1 was subretinally delivered in wildtype animals to lower protein synthesis rates. 4ebp1/2 were knocked out in rd16 mice. Results: Anisomycin treatment lowered retinal translation rates, accelerated retinal degeneration in rd16 mice, and initiated cell death in the retinas of C57BL/6J mice. AAV-mediated transfer of constitutively active 4ebp1-4A into the subretinal space of wildtype animals inhibited protein synthesis, and led to reduced electroretinography amplitudes and fewer ONL nuclei. Finally, we report that restoring protein synthesis rates by knocking out 4ebp1/2 was associated with an approximately 2-fold increase in rhodopsin levels and a delay in retinal degeneration in rd16 mice. Conclusions: Our study indicates that protein synthesis inhibition is likely not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but may be harmful to degenerating retinas, and that restoring protein synthesis may have therapeutic potential in delaying the progression of retinal degeneration.


Assuntos
Biossíntese de Proteínas/fisiologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Anisomicina/farmacologia , Proteínas de Ciclo Celular/genética , Morte Celular , Eletrorretinografia , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica/fisiologia , Marcação In Situ das Extremidades Cortadas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parvovirinae/genética , Inibidores da Síntese de Proteínas/farmacologia , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Transfecção
7.
Nat Protoc ; 14(12): 3333-3365, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31685960

RESUMO

Proteins are continually produced and degraded, to avoid the accumulation of old or damaged molecules and to maintain the efficiency of physiological processes. Despite its importance, protein turnover has been difficult to measure in vivo. Previous approaches to evaluating turnover in vivo have required custom labeling approaches, involved complex mass spectrometry (MS) analyses, or used comparative strategies that do not allow direct quantitative measurements. Here, we describe a robust protocol for quantitative proteome turnover analysis in mice that is based on a commercially available diet for stable isotope labeling of amino acids in mammals (SILAM). We start by discussing fundamental concepts of protein turnover, including different methodological approaches. We then cover in detail the practical aspects of metabolic labeling and explain both the experimental and computational steps that must be taken to obtain accurate in vivo results. Finally, we present a simple experimental workflow that enables measurement of precise turnover rates in a time frame of ~4-5 weeks, including the labeling time. We also provide all the scripts needed for the interpretation of the MS results and for comparing turnover across different conditions. Overall, the workflow presented here comprises several improvements in the determination of protein lifetimes with respect to other available methods, including a minimally invasive labeling strategy and a robust interpretation of MS results, thus enhancing reproducibility across laboratories.


Assuntos
Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Aminoácidos/metabolismo , Animais , Marcação por Isótopo/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas/fisiologia , Proteínas/metabolismo , Proteólise , Proteoma/metabolismo , Reprodutibilidade dos Testes , Fluxo de Trabalho
8.
Mol Cell Biol ; 39(24)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31591142

RESUMO

The eukaryotic cytosol contains multiple RNP granules, including P-bodies and stress granules. Three different methods have been used to describe the transcriptome of stress granules or P-bodies, but how these methods compare and how RNA partitioning occurs between P-bodies and stress granules have not been addressed. Here, we compare the analysis of the stress granule transcriptome based on differential centrifugation with and without subsequent stress granule immunopurification. We find that while differential centrifugation alone gives a first approximation of the stress granule transcriptome, this methodology contains nonspecific transcripts that play a confounding role in the interpretation of results. We also immunopurify and compare the RNAs in stress granules and P-bodies under arsenite stress and compare those results to those for the P-body transcriptome described under nonstress conditions. We find that the P-body transcriptome is dominated by poorly translated mRNAs under nonstress conditions, but during arsenite stress, when translation is globally repressed, the P-body transcriptome is very similar to the stress granule transcriptome. This suggests that translation is a dominant factor in targeting mRNAs into both P-bodies and stress granules, and during stress, when most mRNAs are untranslated, the composition of P-bodies reflects this broader translation repression.


Assuntos
Grânulos Citoplasmáticos/genética , Perfilação da Expressão Gênica/métodos , Estabilidade de RNA/genética , Linhagem Celular Tumoral , Citosol/metabolismo , Células Eucarióticas , Humanos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Estresse Fisiológico/genética , Transcriptoma/genética
10.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527121

RESUMO

There are two major antigenic forms of Shiga toxin (Stx), Stx1 and Stx2, which bind the same receptor and act on the same target but nonetheless differ in potency. Stx1a is more toxic to cultured cells, but Stx2 subtypes are more potent in animal models. To understand this phenomenon in cultured cells, we used a system that combines flow cytometry with a fluorescent reporter to monitor the Stx-induced inhibition of protein synthesis in single cells. We observed that Vero cells intoxicated with Stx1a behave differently than those intoxicated with Stx2 subtypes: cells challenged with Stx1a exhibited a population-wide loss of protein synthesis, while cells exposed to Stx2a or Stx2c exhibited a dose-dependent bimodal response in which one subpopulation of cells was unaffected (i.e., no loss of protein synthesis). Cells challenged with a hybrid toxin containing the catalytic subunit of Stx1a and the cell-binding subunit of Stx2a also exhibited a bimodal response to intoxication, while cells challenged with a hybrid toxin containing the catalytic subunit of Stx2a and the cell-binding subunit of Stx1a exhibited a population-wide loss of protein synthesis. Other experiments further supported a primary role for the subtype of the B subunit in the outcome of host-Stx interactions. Our collective observations indicate that the bimodal response to Stx2 subtypes is due to relatively weak binding between Stx2 and the host cell that reduces the total functional pool of Stx2 in comparison to that of Stx1a. This explains, in part, the molecular basis for the differential cellular toxicity between Stx1a and Stx2 subtypes.


Assuntos
Biossíntese de Proteínas/fisiologia , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Animais , Domínio Catalítico/genética , Linhagem Celular , Infecções por Escherichia coli/patologia , Citometria de Fluxo , Ligação Proteica/fisiologia , Toxina Shiga I/imunologia , Toxina Shiga II/imunologia , Células Vero
11.
Nat Commun ; 10(1): 4006, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488843

RESUMO

The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.


Assuntos
Archaea/genética , Bactérias/genética , Genes Arqueais/genética , Genes Bacterianos/genética , Iniciação Traducional da Cadeia Peptídica/fisiologia , Terminação Traducional da Cadeia Peptídica/fisiologia , Biossíntese de Proteínas/fisiologia , Archaea/metabolismo , Bactérias/metabolismo , Sequência de Bases , Códon de Iniciação , Escherichia coli/genética , Homologia de Genes , Genes Reporter , Fases de Leitura Aberta/genética , RNA Mensageiro , Regiões Terminadoras Genéticas
12.
Nutrients ; 11(8)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394788

RESUMO

Plant-sourced proteins offer environmental and health benefits, and research increasingly includes them in study formulas. However, plant-based proteins have less of an anabolic effect than animal proteins due to their lower digestibility, lower essential amino acid content (especially leucine), and deficiency in other essential amino acids, such as sulfur amino acids or lysine. Thus, plant amino acids are directed toward oxidation rather than used for muscle protein synthesis. In this review, we evaluate the ability of plant- versus animal-based proteins to help maintain skeletal muscle mass in healthy and especially older people and examine different nutritional strategies for improving the anabolic properties of plant-based proteins. Among these strategies, increasing protein intake has led to a positive acute postprandial muscle protein synthesis response and even positive long-term improvement in lean mass. Increasing the quality of protein intake by improving amino acid composition could also compensate for the lower anabolic potential of plant-based proteins. We evaluated and discussed four nutritional strategies for improving the amino acid composition of plant-based proteins: fortifying plant-based proteins with specific essential amino acids, selective breeding, blending several plant protein sources, and blending plant with animal-based protein sources. These nutritional approaches need to be profoundly examined in older individuals in order to optimize protein intake for this population who require a high-quality food protein intake to mitigate age-related muscle loss.


Assuntos
Anabolizantes , Proteínas na Dieta/administração & dosagem , Proteínas Musculares/administração & dosagem , Músculo Esquelético/fisiologia , Proteínas de Plantas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminoácidos/análise , Animais , Proteínas na Dieta/farmacologia , Digestão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Fenômenos Fisiológicos da Nutrição , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Biossíntese de Proteínas/fisiologia
13.
Nat Commun ; 10(1): 3180, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320634

RESUMO

The effects of cell-to-cell variation (noise) in gene expression have proven difficult to quantify because of the mechanistic coupling of noise to mean expression. To independently quantify the effects of changes in mean expression and noise we determine the fitness landscapes in mean-noise expression space for 33 genes in yeast. For most genes, short-lived (noise) deviations away from the expression optimum are nearly as detrimental as sustained (mean) deviations. Fitness landscapes can be classified by a combination of each gene's sensitivity to protein shortage or surplus. We use this classification to explore evolutionary scenarios for gene expression and find that certain landscape topologies can break the mechanistic coupling of mean and noise, thus promoting independent optimization of both properties. These results demonstrate that noise is detrimental for many genes and reveal non-trivial consequences of mean-noise-fitness topologies for the evolution of gene expression systems.


Assuntos
Regulação Fúngica da Expressão Gênica/genética , Expressão Gênica/genética , Biossíntese de Proteínas/fisiologia , Saccharomyces cerevisiae/genética , Aptidão Genética/genética , Modelos Genéticos , Análise de Componente Principal , Biossíntese de Proteínas/genética
14.
Int J Mol Sci ; 20(14)2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31323794

RESUMO

Impairments in translation have been increasingly implicated in the pathogenesis and progression of multiple neurodegenerative diseases. Assessing the spatiotemporal dynamics of translation in the context of disease is a major challenge. Recent developments in proteomic analyses have enabled the resolution of nascent peptides in a short timescale on the order of minutes. In addition, a quantitative analysis of translation has progressed in vivo, showing remarkable potential for coupling these techniques with cognitive and behavioral outcomes. Here, we review these modern approaches to measure changes in translation and ribosomal function with a specific focus on current applications in the mammalian brain and in the study of neurodegenerative diseases.


Assuntos
Proteômica/métodos , Ribossomos/metabolismo , Animais , Humanos , Doenças Neurodegenerativas/metabolismo , Biossíntese de Proteínas/fisiologia
15.
Respir Res ; 20(1): 148, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299951

RESUMO

Idiopathic pulmonary fibrosis (IPF) is characterised by excessive extracellular matrix (ECM) deposition and remodelling. Measuring this activity provides an opportunity to develop tools capable of identifying individuals at-risk of progression. Longitudinal change in markers of ECM synthesis was assessed in 145 newly-diagnosed individuals with IPF.Serum levels of collagen synthesis neoepitopes, PRO-C3 and PRO-C6 (collagen type 3 and 6), were elevated in IPF compared with controls at baseline, and progressive disease versus stable disease during follow up, (PRO-C3 p < 0.001; PRO-C6 p = 0.029). Assessment of rate of change in neoepitope levels from baseline to 3 months (defined as 'slope to month 3': HIGH slope, slope > 0 vs. LOW slope, slope < =0) demonstrated no relationship with mortality for these markers (PRO-C3 (HR 1.62, p = 0.080); PINP (HR 0.76, p = 0.309); PRO-C6 (HR 1.14, p = 0.628)). As previously reported, rising concentrations of collagen degradation markers C1M, C3M, C6M and CRPM were associated with an increased risk of overall mortality (HR = 1.84, CI 1.03-3.27, p = 0.038, HR = 2.44, CI 1.39-4.31, p = 0.002; HR = 2.19, CI 1.25-3.82, p = 0.006; HR = 2.13 CI 1.21-3.75, p = 0.009 respectively).Elevated levels of PRO-C3 and PRO-C6 associate with IPF disease progression. Collagen synthesis and degradation biomarkers have the potential to enhance clinical trials in IPF and may inform prognostic assessment and therapeutic decision making in the clinic.


Assuntos
Colágeno/biossíntese , Colágeno/sangue , Progressão da Doença , Fibrose Pulmonar Idiopática/sangue , Fibrose Pulmonar Idiopática/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Coortes , Humanos , Estudos Longitudinais , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Biossíntese de Proteínas/fisiologia
16.
Nat Cell Biol ; 21(7): 889-899, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31263264

RESUMO

The c-Myc oncogene drives malignant progression and induces robust anabolic and proliferative programmes leading to intrinsic stress. The mechanisms enabling adaptation to MYC-induced stress are not fully understood. Here we reveal an essential role for activating transcription factor 4 (ATF4) in survival following MYC activation. MYC upregulates ATF4 by activating general control nonderepressible 2 (GCN2) kinase through uncharged transfer RNAs. Subsequently, ATF4 co-occupies promoter regions of over 30 MYC-target genes, primarily those regulating amino acid and protein synthesis, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation. 4E-BP1 relieves MYC-induced proteotoxic stress and is essential to balance protein synthesis. 4E-BP1 activity is negatively regulated by mammalian target of rapamycin complex 1 (mTORC1)-dependent phosphorylation and inhibition of mTORC1 signalling rescues ATF4-deficient cells from MYC-induced endoplasmic reticulum stress. Acute deletion of ATF4 significantly delays MYC-driven tumour progression and increases survival in mouse models. Our results establish ATF4 as a cellular rheostat of MYC activity, which ensures that enhanced translation rates are compatible with survival and tumour progression.


Assuntos
Fator 4 Ativador da Transcrição/genética , Genes myc/genética , Ativação Transcricional/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Estresse do Retículo Endoplasmático/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Transgênicos , Fosfoproteínas/genética , Fosforilação , Biossíntese de Proteínas/fisiologia , Serina-Treonina Quinases TOR/metabolismo
17.
Nat Commun ; 10(1): 2709, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221966

RESUMO

Protein folding can begin co-translationally. Due to the difference in timescale between folding and synthesis, co-translational folding is thought to occur at equilibrium for fast-folding domains. In this scenario, the folding kinetics of stalled ribosome-bound nascent chains should match the folding of nascent chains in real time. To test if this assumption is true, we compare the folding of a ribosome-bound, multi-domain calcium-binding protein stalled at different points in translation with the nascent chain as is it being synthesized in real-time, via optical tweezers. On stalled ribosomes, a misfolded state forms rapidly (1.5 s). However, during translation, this state is only attained after a long delay (63 s), indicating that, unexpectedly, the growing polypeptide is not equilibrated with its ensemble of accessible conformations. Slow equilibration on the ribosome can delay premature folding until adequate sequence is available and/or allow time for chaperone binding, thus promoting productive folding.


Assuntos
Modelos Moleculares , Biossíntese de Proteínas/fisiologia , Dobramento de Proteína , Proteínas de Bactérias/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Chaperonas Moleculares/metabolismo , Pinças Ópticas , Peptídeos/metabolismo , Domínios Proteicos/fisiologia , Ribossomos/metabolismo , Fatores de Tempo
18.
PLoS Biol ; 17(6): e3000297, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199786

RESUMO

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.


Assuntos
DNA-Citosina Metilases/metabolismo , Metiltransferases/metabolismo , Animais , Linhagem Celular , Citosina/metabolismo , Metilação de DNA/fisiologia , DNA-Citosina Metilases/fisiologia , Humanos , Camundongos , Estresse Oxidativo/fisiologia , Biossíntese de Proteínas/fisiologia , RNA/metabolismo , RNA de Transferência/metabolismo
19.
Nat Commun ; 10(1): 2640, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201334

RESUMO

One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNAi.


Assuntos
Fator de Iniciação 2B em Eucariotos/ultraestrutura , Fator de Iniciação 2 em Eucariotos/ultraestrutura , Biossíntese de Proteínas/fisiologia , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Guanosina Difosfato/metabolismo , Modelos Moleculares , Fosforilação/fisiologia , Ligação Proteica/fisiologia , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Metionina/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
20.
Exp Hematol ; 75: 11-20, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31154069

RESUMO

Translational regulation plays a critical role in erythropoiesis, as it reflects the translational needs of enucleated mature erythroid cells in the absence of transcription and the large translational demands of balanced globin chain synthesis during erythroid maturation. In addition, red blood cells need to respond quickly to changes in their environment and the demands of the organism. Translational regulation occurs at several levels in erythroid cells, including the differential utilization of upstream open reading frames during differentiation and in response to signaling and the employment of RNA-binding proteins in an erythroid cell-specific fashion. Translation initiation is a critical juncture for translational regulation in response to environmental signals such as heme and iron availability, whereas regulatory mechanisms for ribosome recycling are consistent with recent observations highlighting the importance of maintaining adequate ribosome levels in differentiating erythroid cells. Translational deregulation in erythroid cells leads to disease associated with ineffective erythropoiesis, further highlighting the pivotal role translational regulation in erythropoiesis plays in human physiology and homeostasis. Overall, erythropoiesis has served as a unique model that has provided invaluable insight into translational regulation.


Assuntos
Diferenciação Celular/fisiologia , Células Eritroides/metabolismo , Eritropoese/fisiologia , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Células Eritroides/citologia , Heme/metabolismo , Humanos , Ferro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA