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1.
Nutrients ; 13(8)2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34444950

RESUMO

The purpose of this research was to investigate the prophylactic effects of glutamine on muscle protein synthesis and degradation in rats with ethanol-induced liver injury. For the first 2 weeks, Wistar rats were divided into two groups and fed a control (n = 16) or glutamine-containing diet (n = 24). For the following 6 weeks, rats fed the control diet were further divided into two groups (n = 8 per group) according to whether their diet contained no ethanol (CC) or did contain ethanol (CE). Rats fed the glutamine-containing diet were also further divided into three groups (n = 8 per group), including a GG group (glutamine-containing diet without ethanol), GE group (control diet with ethanol), and GEG group (glutamine-containing diet with ethanol). After 6 weeks, results showed that hepatic fatty change, inflammation, altered liver function, and hyperammonemia had occurred in the CE group, but these were attenuated in the GE and GEG groups. Elevated intestinal permeability and a higher plasma endotoxin level were observed in the CE group, but both were lower in the GE and GEG groups. The level of a protein synthesis marker (p70S6K) was reduced in the CE group but was higher in both the GE and GEG groups. In conclusion, glutamine supplementation might elevate muscle protein synthesis by improving intestinal health and ameliorating liver damage in rats with chronic ethanol intake.


Assuntos
Glutamina/administração & dosagem , Hepatopatias Alcoólicas/prevenção & controle , Proteínas Musculares/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Etanol , Inflamação , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Hepatopatias Alcoólicas/etiologia , Ratos , Ratos Wistar
2.
Nat Commun ; 12(1): 5094, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429433

RESUMO

Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.


Assuntos
Cicloeximida/farmacologia , Ribossomos/química , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Animais , Viés , Códon/metabolismo , Células HEK293 , Humanos , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie
3.
Nutrients ; 13(7)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34371831

RESUMO

Angiotensin converting enzyme 2 (ACE2) is a key entry point of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus known to induce Coronavirus disease 2019 (COVID-19). We have recently outlined a concept to reduce ACE2 expression by the administration of glycyrrhizin, a component of Glycyrrhiza glabra extract, via its inhibitory activity on 11beta hydroxysteroid dehydrogenase type 2 (11betaHSD2) and resulting activation of mineralocorticoid receptor (MR). We hypothesized that in organs such as the ileum, which co-express 11betaHSD2, MR and ACE2, the expression of ACE2 would be suppressed. We studied organ tissues from an experiment originally designed to address the effects of Glycyrrhiza glabra extract on stress response. Male Sprague Dawley rats were left undisturbed or exposed to chronic mild stress for five weeks. For the last two weeks, animals continued with a placebo diet or received a diet containing extract of Glycyrrhiza glabra root at a dose of 150 mg/kg of body weight/day. Quantitative PCR measurements showed a significant decrease in gene expression of ACE2 in the small intestine of rats fed with diet containing Glycyrrhiza glabra extract. This effect was independent of the stress condition and failed to be observed in non-target tissues, namely the heart and the brain cortex. In the small intestine we also confirmed the reduction of ACE2 at the protein level. Present findings provide evidence to support the hypothesis that Glycyrrhiza glabra extract may reduce an entry point of SARS-CoV-2. Whether this phenomenon, when confirmed in additional studies, is linked to the susceptibility of cells to the virus requires further studies.


Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , COVID-19/tratamento farmacológico , Suplementos Nutricionais , Glycyrrhiza , Extratos Vegetais/uso terapêutico , Biossíntese de Proteínas/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Ácido Glicirrízico/administração & dosagem , Ácido Glicirrízico/uso terapêutico , Masculino , Extratos Vegetais/administração & dosagem , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley
4.
Nat Commun ; 12(1): 4920, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389715

RESUMO

Malignant mesothelioma (MpM) is an aggressive, invariably fatal tumour that is causally linked with asbestos exposure. The disease primarily results from loss of tumour suppressor gene function and there are no 'druggable' driver oncogenes associated with MpM. To identify opportunities for management of this disease we have carried out polysome profiling to define the MpM translatome. We show that in MpM there is a selective increase in the translation of mRNAs encoding proteins required for ribosome assembly and mitochondrial biogenesis. This results in an enhanced rate of mRNA translation, abnormal mitochondrial morphology and oxygen consumption, and a reprogramming of metabolic outputs. These alterations delimit the cellular capacity for protein biosynthesis, accelerate growth and drive disease progression. Importantly, we show that inhibition of mRNA translation, particularly through combined pharmacological targeting of mTORC1 and 2, reverses these changes and inhibits malignant cell growth in vitro and in ex-vivo tumour tissue from patients with end-stage disease. Critically, we show that these pharmacological interventions prolong survival in animal models of asbestos-induced mesothelioma, providing the basis for a targeted, viable therapeutic option for patients with this incurable disease.


Assuntos
Mesotelioma Maligno/genética , Oncogenes/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Animais , Asbestos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Mesotelioma Maligno/induzido quimicamente , Mesotelioma Maligno/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Naftiridinas/farmacologia , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas
5.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198827

RESUMO

The objective of this study was to investigate molecular mechanisms underlying the ability of carnosic acid to attenuate an early increase in reactive oxygen species (ROS) levels during MDI-induced adipocyte differentiation. The levels of superoxide anion and ROS were determined using dihydroethidium (DHE) and 2'-7'-dichlorofluorescin diacetate (DCFH-DA), respectively. Both superoxide anion and ROS levels peaked on the second day of differentiation. They were suppressed by carnosic acid. Carnosic acid attenuates the translation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4), p47phox, and p22phox, and the phosphorylation of nuclear factor-kappa B (NF-κB) and NF-κB inhibitor (IkBa). The translocation of NF-κB into the nucleus was also decreased by carnosic acid. In addition, carnosic acid increased the translation of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCSc), and glutathione S-transferase (GST) and both the translation and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, these results indicate that carnosic acid could down-regulate ROS level in an early stage of MPI-induced adipocyte differentiation by attenuating ROS generation through suppression of NF-κB-mediated translation of Nox4 enzyme and increasing ROS neutralization through induction of Nrf2-mediated translation of phase II antioxidant enzymes such as HO-1, γ-GCS, and GST, leading to its anti-adipogenetic effect.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Abietanos/farmacologia , DNA Helicases/genética , Heme Oxigenase-1/genética , Proteínas de Membrana/genética , NADPH Oxidase 4/genética , Inibidor de NF-kappaB alfa/genética , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Antioxidantes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Grupo dos Citocromos b/genética , Etídio/análogos & derivados , Etídio/farmacologia , Fluoresceínas/farmacologia , Glutationa Transferase/genética , Camundongos , NADPH Oxidases/genética , Biossíntese de Proteínas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
6.
Nat Commun ; 12(1): 4466, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294725

RESUMO

Macrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.


Assuntos
Antibacterianos/farmacologia , Cetolídeos/farmacologia , Macrolídeos/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Antibacterianos/química , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Resistência Microbiana a Medicamentos/genética , Eritromicina/química , Eritromicina/farmacologia , Genes Bacterianos , Cetolídeos/química , Cetolídeos/farmacocinética , Macrolídeos/química , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Insercional , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , Ribossomos/efeitos dos fármacos
7.
Nature ; 596(7871): 291-295, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321659

RESUMO

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Edição de Genes/métodos , Terapia Genética/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Eritropoetina/biossíntese , Eritropoetina/genética , Eritropoetina/metabolismo , Éxons/genética , Feminino , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular Espinal/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Progranulinas/biossíntese , Progranulinas/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
8.
Mol Cell ; 81(15): 3160-3170.e9, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34174184

RESUMO

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.


Assuntos
Toxinas Bacterianas/metabolismo , Guanosina Pentafosfato/metabolismo , Ligases/metabolismo , RNA de Transferência/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Bacilos Gram-Positivos Asporogênicos/química , Bacilos Gram-Positivos Asporogênicos/metabolismo , Guanosina Pentafosfato/química , Ligases/química , Ligases/genética , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Pirofosfatases , Ribossomos/metabolismo
9.
Nucleic Acids Res ; 49(12): 6880-6892, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34125898

RESUMO

How aminoglycoside antibiotics limit bacterial growth and viability is not clearly understood. Here we employ fast kinetics to reveal the molecular mechanism of action of a clinically used, new-generation, semisynthetic aminoglycoside Arbekacin (ABK), which is designed to avoid enzyme-mediated deactivation common to other aminoglycosides. Our results portray complete picture of ABK inhibition of bacterial translation with precise quantitative characterizations. We find that ABK inhibits different steps of translation in nanomolar to micromolar concentrations by imparting pleotropic effects. ABK binding stalls elongating ribosomes to a state, which is unfavorable for EF-G binding. This prolongs individual translocation step from ∼50 ms to at least 2 s; the mean time of translocation increases inversely with EF-G concentration. ABK also inhibits translation termination by obstructing RF1/RF2 binding to the ribosome. Furthermore, ABK decreases accuracy of mRNA decoding (UUC vs. CUC) by ∼80 000 fold, causing aberrant protein production. Importantly, translocation and termination events cannot be completely stopped even with high ABK concentration. Extrapolating our kinetic model of ABK action, we postulate that aminoglycosides impose bacteriostatic effect mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors.


Assuntos
Antibacterianos/farmacologia , Dibecacina/análogos & derivados , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos , Antibacterianos/química , Dibecacina/química , Dibecacina/farmacologia , Cinética , Fator G para Elongação de Peptídeos/antagonistas & inibidores , Fatores de Terminação de Peptídeos/antagonistas & inibidores , Peptídeos/metabolismo , Inibidores da Síntese de Proteínas/química , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/metabolismo
10.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069640

RESUMO

Bacteria have evolved an array of mechanisms enabling them to resist the inhibitory effect of antibiotics, a significant proportion of which target the ribosome. Indeed, resistance mechanisms have been identified for nearly every antibiotic that is currently used in clinical practice. With the ever-increasing list of multi-drug-resistant pathogens and very few novel antibiotics in the pharmaceutical pipeline, treatable infections are likely to become life-threatening once again. Most of the prevalent resistance mechanisms are well understood and their clinical significance is recognized. In contrast, ribosome protection protein-mediated resistance has flown under the radar for a long time and has been considered a minor factor in the clinical setting. Not until the recent discovery of the ATP-binding cassette family F protein-mediated resistance in an extensive list of human pathogens has the significance of ribosome protection proteins been truly appreciated. Understanding the underlying resistance mechanism has the potential to guide the development of novel therapeutic approaches to evade or overcome the resistance. In this review, we discuss the latest developments regarding ribosome protection proteins focusing on the current antimicrobial arsenal and pharmaceutical pipeline as well as potential implications for the future of fighting bacterial infections in the time of "superbugs."


Assuntos
Resistência Microbiana a Medicamentos/fisiologia , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Modelos Moleculares , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Ribossômicas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos
11.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34110129

RESUMO

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Ebolavirus/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Alanina/genética , Alanina/metabolismo , Antivirais/metabolismo , Pareamento de Bases , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/metabolismo , Modelos Moleculares , Biossíntese de Proteínas/efeitos dos fármacos , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
12.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34066907

RESUMO

Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, translational readthrough of premature termination codons (PTC-readthrough) induced by aminoglycosides has been proposed as an effective way of restoring functional protein expression and reducing disease symptoms. However, variable outcomes of pre-clinical trials and toxicity associated with long-term use of aminoglycosides prompt the search for other compounds that might overcome these problems. Because a high proportion of PCD-causing variants are nonsense mutations, readthrough therapies are an attractive option. We tested a group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007), collectively referred to as non-aminoglycosides (NAGs). We investigated their PTC-readthrough efficiency in six PTC mutations found in Polish PCD patients, in the context of cell and cilia health, and in comparison to the previously tested aminoglycosides. The NAGs did not compromise the viability of the primary nasal respiratory epithelial cells, and the ciliary beat frequency was retained, similar to what was observed for gentamicin. In HEK293 cells transfected with six PTC-containing inserts, the tested compounds stimulated PTC-readthrough but with lower efficiency than aminoglycosides. The study allowed us to select compounds with minimal negative impact on cell viability and function but still the potential to induce PTC-readthrough.


Assuntos
Aminoglicosídeos/farmacologia , Transtornos da Motilidade Ciliar/genética , Códon sem Sentido/genética , Mutação/genética , Biossíntese de Proteínas/genética , Morte Celular/efeitos dos fármacos , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Nariz/patologia , Biossíntese de Proteínas/efeitos dos fármacos
13.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064311

RESUMO

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.


Assuntos
Região CA1 Hipocampal/metabolismo , Região CA3 Hipocampal/metabolismo , Calcineurina/genética , Potenciação de Longa Duração/genética , Potenciais Sinápticos/genética , Animais , Anisomicina/farmacologia , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/efeitos dos fármacos , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/efeitos dos fármacos , Calcineurina/metabolismo , Inibidores de Calcineurina/farmacologia , Cicloeximida/farmacologia , Ciclosporina/farmacologia , Giro Denteado/citologia , Giro Denteado/efeitos dos fármacos , Giro Denteado/metabolismo , Regulação da Expressão Gênica , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Microtomia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Óxido Nítrico/química , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Wistar , S-Nitroso-N-Acetilpenicilamina/química , S-Nitroso-N-Acetilpenicilamina/farmacologia , Potenciais Sinápticos/efeitos dos fármacos , Técnicas de Cultura de Tecidos
14.
PLoS Biol ; 19(5): e3001263, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34033645

RESUMO

We here conducted an image-based chemical screen to evaluate how medically approved drugs, as well as drugs that are currently under development, influence overall translation levels. None of the compounds up-regulated translation, which could be due to the screen being performed in cancer cells grown in full media where translation is already present at very high levels. Regarding translation down-regulators, and consistent with current knowledge, inhibitors of the mechanistic target of rapamycin (mTOR) signaling pathway were the most represented class. In addition, we identified that inhibitors of sphingosine kinases (SPHKs) also reduce mRNA translation levels independently of mTOR. Mechanistically, this is explained by an effect of the compounds on the membranes of the endoplasmic reticulum (ER), which activates the integrated stress response (ISR) and contributes to the toxicity of SPHK inhibitors. Surprisingly, the toxicity and activation of the ISR triggered by 2 independent SPHK inhibitors, SKI-II and ABC294640, the latter in clinical trials, are also observed in cells lacking SPHK1 and SPHK2. In summary, our study provides a useful resource on the effects of medically used drugs on translation, identified compounds capable of reducing translation independently of mTOR and has revealed that the cytotoxic properties of SPHK inhibitors being developed as anticancer agents are independent of SPHKs.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Biossíntese de Proteínas/fisiologia , Animais , Linhagem Celular , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Lisofosfolipídeos/metabolismo , Espectrometria de Massas/métodos , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Esfingosina/metabolismo
15.
J Biol Chem ; 297(1): 100839, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34051232

RESUMO

Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic ß-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human ß-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human ß-cells, it plays a major role at the translational level.


Assuntos
Metabolismo Energético/genética , Glucose/farmacologia , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas/genética , Linhagem Celular , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pró-Proteína Convertase 1/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
16.
Biomed Pharmacother ; 140: 111765, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34058438

RESUMO

Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase ß (IKKß). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E2 (PGE2), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically.


Assuntos
Inibidores da Angiogênese/farmacologia , Anti-Inflamatórios/farmacologia , Benzopiranos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Selectina E/genética , Selectina E/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Camundongos , NF-kappa B/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
17.
PLoS Biol ; 19(5): e3001221, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33939688

RESUMO

Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.


Assuntos
Aminoglicosídeos/farmacologia , Códon sem Sentido/genética , Ribossomos/metabolismo , Aminoglicosídeos/metabolismo , Aminoglicosídeos/fisiologia , Linhagem Celular , Quimiocinas CXC/efeitos dos fármacos , Quimiocinas CXC/metabolismo , Códon sem Sentido/metabolismo , Códon de Terminação , Gentamicinas/farmacologia , Humanos , Mutação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas , Ribossomos/efeitos dos fármacos
18.
Nat Commun ; 12(1): 2803, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990576

RESUMO

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.


Assuntos
Antibacterianos/farmacologia , Macrolídeos/farmacologia , Ribossomos/efeitos dos fármacos , Antibacterianos/química , Sítios de Ligação , Microscopia Crioeletrônica , Células Eucarióticas/efeitos dos fármacos , Células Eucarióticas/metabolismo , Humanos , Macrolídeos/química , Modelos Moleculares , Mutação , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/farmacologia , RNA Fúngico/genética , RNA Ribossômico/genética , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Relação Estrutura-Atividade
19.
Nat Commun ; 12(1): 2507, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947863

RESUMO

Notch1 is a crucial oncogenic driver in T-cell acute lymphoblastic leukemia (T-ALL), making it an attractive therapeutic target. However, the success of targeted therapy using γ-secretase inhibitors (GSIs), small molecules blocking Notch cleavage and subsequent activation, has been limited due to development of resistance, thus restricting its clinical efficacy. Here, we systematically compare GSI resistant and sensitive cell states by quantitative mass spectrometry-based phosphoproteomics, using complementary models of resistance, including T-ALL patient-derived xenografts (PDX) models. Our datasets reveal common mechanisms of GSI resistance, including a distinct kinase signature that involves protein kinase C delta. We demonstrate that the PKC inhibitor sotrastaurin enhances the anti-leukemic activity of GSI in PDX models and completely abrogates the development of acquired GSI resistance in vitro. Overall, we highlight the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Oligopeptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteína Quinase C/metabolismo , Receptor Notch1/antagonistas & inibidores , Acetofenonas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Antineoplásicos/uso terapêutico , Benzopiranos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Cromatografia Líquida de Alta Pressão , Resistencia a Medicamentos Antineoplásicos/genética , Ontologia Genética , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos NOD , Fosforilação , Análise Serial de Proteínas , Biossíntese de Proteínas/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteínas Quinases/metabolismo , Proteômica , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em Tandem , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Nat Commun ; 12(1): 2589, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972524

RESUMO

Patients with Tuberous Sclerosis Complex (TSC) show aberrant wiring of neuronal connections formed during development which may contribute to symptoms of TSC, such as intellectual disabilities, autism, and epilepsy. Yet models examining the molecular basis for axonal guidance defects in developing human neurons have not been developed. Here, we generate human induced pluripotent stem cell (hiPSC) lines from a patient with TSC and genetically engineer counterparts and isogenic controls. By differentiating hiPSCs, we show that control neurons respond to canonical guidance cues as predicted. Conversely, neurons with heterozygous loss of TSC2 exhibit reduced responses to several repulsive cues and defective axon guidance. While TSC2 is a known key negative regulator of MTOR-dependent protein synthesis, we find that TSC2 signaled through MTOR-independent RHOA in growth cones. Our results suggest that neural network connectivity defects in patients with TSC may result from defects in RHOA-mediated regulation of cytoskeletal dynamics during neuronal development.


Assuntos
Orientação de Axônios/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Rede Nervosa/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adolescente , Orientação de Axônios/efeitos dos fármacos , Biópsia , Sistemas CRISPR-Cas , Linhagem Celular , Efrinas/farmacologia , Transferência Ressonante de Energia de Fluorescência , Haploinsuficiência , Heterozigoto , Humanos , Masculino , Miosinas/metabolismo , Rede Nervosa/patologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Organoides/citologia , Organoides/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética
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