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1.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638549

RESUMO

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Antifúngicos/farmacologia , Cicloeximida/farmacologia , Endocitose/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação/efeitos dos fármacos
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.
Sci Rep ; 11(1): 14154, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34238994

RESUMO

Congenital heart defects, one of the most common birth defects, affect approximately 1% of live birth globally and remain the leading cause of infant mortality in developed countries. Utilizing the pathogenicity score and inheritance mode from whole exome sequencing results, a heterozygous mutation (NM_001278939.1: c.1939G>T, p.Gly647Ter) in elastin (ELN) was identified among 6,440 variants in a female proband born with an atrial septal defect accompanied by pulmonary artery stenosis. Results of RT-PCR showed that the mutation (NM_001278939.1: c.1939G>T, p.Gly647Ter) did not affect the expression levels of ELN mRNA but increased protein level. The content of ELN truncate (functional component) was significantly lower in both the intracellular and extracellular compartments after mutation. These results indicate that the ELN mutation (NM_001278939.1: c.1939G>T, p.Gly647Ter) affected the protein truncate, which may be a functional component of ELN and play crucial roles for this pedigree. Here we report of an ELN heterozygous variant associated with congenital heart disease accompanied with pulmonary artery stenosis, which is less common. Based on our results, we speculate that this may be the main molecular mechanism underlying the mutation-led functional changes, and propose that the decrease of ELN protein level may cause this pedigree vascular abnormality, especially pulmonary artery stenosis, and reinforce the view that ELN insufficiency is the primary cause of these vascular lesions. This may be the main molecular mechanism underlying the mutation-led functional changes. Thus, systematic analysis not only enables us to better understand the etiology of this disease but also contributes to clinical and prenatal diagnosis.


Assuntos
Elastina/genética , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/genética , Mutação/genética , Estenose de Artéria Pulmonar/complicações , Estenose de Artéria Pulmonar/genética , Sequência de Bases , Cicloeximida/farmacologia , Análise Mutacional de DNA , Eletrocardiografia , Feminino , Células HEK293 , Cardiopatias Congênitas/diagnóstico por imagem , Humanos , Lactente , Masculino , Linhagem , Estenose de Artéria Pulmonar/diagnóstico por imagem , Sequenciamento Completo do Exoma
4.
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
5.
Development ; 148(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34015092

RESUMO

Upon the stimulation of extracellular cues, a significant number of proteins are synthesized distally along the axon. Although local protein synthesis is crucial for various stages throughout neuronal development, its involvement in presynaptic differentiation at developing neuromuscular junctions remains unknown. By using axon severing and microfluidic chamber assays, we first showed that treatment of a protein synthesis inhibitor, cycloheximide, inhibits agrin-induced presynaptic differentiation in cultured Xenopus spinal neurons. Newly synthesized proteins are prominently detected, as revealed by the staining of click-reactive cell-permeable puromycin analog O-propargyl-puromycin, at agrin bead-neurite contacts involving the mTOR/4E-BP1 pathway. Next, live-cell time-lapse imaging demonstrated the local capturing and immobilization of ribonucleoprotein granules upon agrin bead stimulation. Given that our recent study reported the roles of membrane-type 1 matrix metalloproteinase (MT1-MMP) in agrin-induced presynaptic differentiation, here we further showed that MT1-MMP mRNA is spatially enriched and locally translated at sites induced by agrin beads. Taken together, this study reveals an essential role for axonal MT1-MMP translation, on top of the well-recognized long-range transport of MT1-MMP proteins synthesized from neuronal cell bodies, in mediating agrin-induced presynaptic differentiation.


Assuntos
Agrina/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Neurogênese/fisiologia , Biossíntese de Proteínas/fisiologia , Xenopus laevis/embriologia , Animais , Axônios/metabolismo , Células Cultivadas , Cicloeximida/farmacologia , Metaloproteinase 14 da Matriz/genética , Microfluídica/métodos , Neurogênese/efeitos dos fármacos , Junção Neuromuscular/embriologia , Terminações Pré-Sinápticas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-34043492

RESUMO

Rhodotorula mucilaginosa is an antagonistic yeast for which our research team has recently reported interesting biocontrol activities against blue mould decay of apples and a strong ability to decrease the patulin concentration in vivo. However, the possible mechanisms of patulin degradation by R. mucilaginosa and the toxicity of patulin degradation products remain unclear. In this study, the effect of R. mucilaginosa on patulin degradation and toxicity of degradation products were investigated, the results showed that viable cells of R. mucilaginosa are essential to patulin degradation. Also, R. mucilaginosa eliminated patulin without adsorbing it through its cell wall. The extracellular metabolites of R. mucilaginosa stimulated by patulin showed little degradation activity for patulin. Cycloheximide addition into the medium significantly decreased the patulin degradation capacity of R. mucilaginosa cells. The main patulin degradation product by R. mucilaginosa was ascladiol, which was proved non-toxic to human hepatoma (HepG2) cells at 0.625-10 g/mL. Furthermore, toxicological analysis using a confocal laser scanning microscope revealed that the degradation product induced cellular apoptosis to a lesser extent than patulin itself. This result offers an innovative method to detoxify patulin and limit the risks of patulin in fruits and vegetables using R. mucilaginosa.


Assuntos
Fungos/metabolismo , Furanos/toxicidade , Patulina/metabolismo , Rhodotorula/metabolismo , Cicloeximida/metabolismo , Aditivos Alimentares/metabolismo , Contaminação de Alimentos , Frutas/microbiologia , Fungos/crescimento & desenvolvimento , Células Hep G2 , Humanos , Malus/microbiologia , Metaboloma , Medição de Risco
7.
J Vis Exp ; (170)2021 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-33900287

RESUMO

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker's yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.


Assuntos
Genoma Fúngico , Genoma Mitocondrial , RNA Mensageiro/genética , RNA Mitocondrial/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Cicloeximida/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Coloração e Rotulagem/métodos
8.
Biochem Biophys Res Commun ; 558: 64-70, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33901925

RESUMO

Long-term potentiation (LTP) and long-term depression (LTD) are key forms of synaptic plasticity in the hippocampus. LTP and LTD are believed to underlie the processes occurring during learning and memory. Search of mechanisms responsible for switching from LTP to LTD and vice versa is an important fundamental task. Protein synthesis blockers (PSB) are widely used in models of memory impairment and LTP suppression. Here, we found that blockade of serine/threonine phosphatases 1 (PP1) and 2A (PP2A) with the specific blockers, calyculin A (CalyA) or okadaic acid (OA), and simultaneous blockade of the protein translation by anisomycin or cycloheximide leads to a switch from PSB-impaired LTP to LTD. PP1/PP2A-dependent LTD was extremely sensitive to the intensity of the test stimuli, whose increase restored the field excitatory postsynaptic potentials (fEPSP) to the values corresponding to control LTP in the non-treated slices. PP1/PP2A blockade affected the basal synaptic transmission, increasing the paired-pulse facilitation (PPF) ratio, and restored the PSB-impaired PPF 3 h after tetanus. Prolonged exposure to anisomycin led to the NO synthesis increase (measured using fluorescent dye) both in the dendrites and somata of CA1, CA3, dentate gyrus (DG) hippocampal layers. OA partially prevented the NO production in the CA1 dendrites, as well in the CA3 and DG somas. Direct measurements of changes in serine/threonine phosphatase (STPP) activity revealed importance of the PP1/PP2A-dependent component in the late LTP phase (L-LTP) in anisomycin-treated slices. Thus, serine/threonine phosphatases PP1/PP2A influence both basal synaptic transmission and stimulation-induced synaptic plasticity.


Assuntos
Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 2/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Animais , Anisomicina/farmacologia , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/fisiologia , Cicloeximida/farmacologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/fisiologia , Masculino , Toxinas Marinhas/farmacologia , Óxido Nítrico/biossíntese , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Ratos , Ratos Wistar
9.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803345

RESUMO

We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 and CREB3L2; however, the level of the full-length CREB3 protein, but not CREB3L2 protein, was not noticeably reduced by the monensin treatment. On the other hand, treatment with tunicamycin (Tm) shifted the molecular weight of the full-length CREB3L2 protein downward but abolished CREB3 protein expression. Thapsigargin (Tg) significantly increased the expression of only full-length CREB3L2 protein concomitant with a slight increase in the level of its cleaved form. Treatment with cycloheximide and MG132 revealed that both endogenous CREB3 and CREB3L2 are proteasome substrates. In addition, kifunensine, an α-mannosidase inhibitor, significantly increased the levels of both full-length forms. Consistent with these findings, cells lacking SEL1L, a crucial ER-associated protein degradation (ERAD) component, showed increased expression of both full-length CREB3 and CREB3L2; however, cycloheximide treatment downregulated full-length CREB3L2 protein expression more rapidly in SEL1L-deficient cells than the full-length CREB3 protein. Finally, we investigated the induction of the expression of several CREB3 and CREB3L2 target genes by Tg and BFA treatments and SEL1L deficiency. In conclusion, this study suggests that both endogenous full-length CREB3 and CREB3L2 are substrates for ER-associated protein degradation but are partially regulated by distinct mechanisms, each of which contributes to unique cellular responses that are distinct from canonical ER signals.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Regulação da Expressão Gênica , Alcaloides/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Cicloeximida/farmacologia , Células HEK293 , Humanos , Leupeptinas/farmacologia , Proteínas/genética , Proteínas/metabolismo
10.
Biochem Biophys Res Commun ; 554: 76-82, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33784509

RESUMO

It has been implied that deregulation of cyclin D1 turnover under stresses can facilitate genomic instability and trigger tumorigenesis. Much focus has been placed on identifying the E3 ligases responsible for mediating cyclin D1 degradation. However, the findings were quite controversial and cell type-dependent. Little is known about how cyclin D1 is regulated in precancerous cells upon DNA damage and which E3 ligases mediate the effects. Here we found cyclin D1 reduction is an early response to DNA damage in immortalized esophageal epithelial cells, with expression dropping to a low level within 1 h after γ-irradiation. Comparison of temporal expression of cyclin D1 upon DNA damage between immortalized NE083-hTERT and NE083-E6E7, the latter being p53/p21-defective, showed that DNA damage-induced rapid cyclin D1 reduction was p53-independent and occurred before p21 accumulation. Overexpression of cyclin D1 in NE083-E6E7 cells could attenuate G0/G1 cell cycle arrest at 1 h after irradiation. Furthermore, rapid reduction of cyclin D1 upon DNA damage was attributed to proteasomal degradation, as evidenced by data showing that proteasomal inhibition by MG132 blocked cyclin D1 reduction while cycloheximide facilitated it. Inhibition of ATM activation and knockdown of E3 ligase adaptor FBX4 reversed cyclin D1 turnover in immortalized NE083-hTERT cells. Further study showed that knockdown of FBX4 facilitated DNA breaks, as indicated by an increase in γ-H2AX foci in esophageal cancer cells. Taken together, the results substantiated a pivotal role of ATM and FBX4 in cyclin D1 proteolysis upon DNA damage in precancerous esophageal epithelial cells, implying that deregulation of the process may contribute to carcinogenesis of esophageal squamous cell carcinoma.


Assuntos
Ciclina D1/metabolismo , Dano ao DNA , Esôfago/metabolismo , Proteínas F-Box/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclina D1/biossíntese , Ciclina D1/genética , Cicloeximida/farmacologia , Regulação para Baixo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Esôfago/efeitos dos fármacos , Esôfago/patologia , Esôfago/efeitos da radiação , Proteínas F-Box/biossíntese , Proteínas F-Box/genética , Raios gama , Humanos , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
11.
Plant Physiol ; 185(2): 519-532, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33721908

RESUMO

The circadian clock coordinates the physiological responses of a biological system to day and night rhythms through complex loops of transcriptional/translational regulation. It can respond to external stimuli and adjust generated circadian oscillations accordingly to maintain an endogenous period close to 24 h. However, the interaction between nutritional status and circadian rhythms in plants is poorly understood. Magnesium (Mg) is essential for numerous biological processes in plants, and its homeostasis is crucial to maintain optimal development and growth. Magnesium deficiency in young Arabidopsis thaliana seedlings increased the period of circadian oscillations of the CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) promoter (pCCA1:LUC) activity and dampened their amplitude under constant light in a dose-dependent manner. Although the circadian period increase caused by Mg deficiency was light dependent, it did not depend on active photosynthesis. Mathematical modeling of the Mg input into the circadian clock reproduced the experimental increase of the circadian period and suggested that Mg is likely to affect global transcription/translation levels rather than a single component of the circadian oscillator. Upon addition of a low dose of cycloheximide to perturb translation, the circadian period increased further under Mg deficiency, which was rescued when sufficient Mg was supplied, supporting the model's prediction. These findings suggest that sufficient Mg supply is required to support proper timekeeping in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Magnésio/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Cicloeximida/farmacologia , Homeostase , Luz , Deficiência de Magnésio , Modelos Teóricos , Regiões Promotoras Genéticas/genética , Plântula/genética , Plântula/fisiologia , Plântula/efeitos da radiação , Fatores de Tempo , Fatores de Transcrição/genética
12.
Biosci Biotechnol Biochem ; 85(5): 1290-1293, 2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33784739

RESUMO

Dihydropyriculol is a major secondary metabolite of Pyricularia oryzae. However, the biological activity of dihydropyriculol has not been reported. Here, we showed that dihydropyriculol has inhibitory activity against Streptomyces griseus. Localization analysis of dihydropyriculol revealed that dihydropyriculol could reach to S. griseus under confrontation culture. These results suggest that dihydropyriculol can be used as a chemical weapon against S. griseus.


Assuntos
Antibacterianos/toxicidade , Ascomicetos/metabolismo , Benzaldeídos/toxicidade , Álcoois Graxos/toxicidade , Streptomyces griseus/efeitos dos fármacos , Toxinas Biológicas/toxicidade , Antibacterianos/biossíntese , Antibiose , Ascomicetos/efeitos dos fármacos , Ascomicetos/patogenicidade , Benzaldeídos/metabolismo , Cicloeximida/farmacologia , Álcoois Graxos/metabolismo , Gentamicinas/farmacologia , Higromicina B/farmacologia , Testes de Sensibilidade Microbiana , Metabolismo Secundário/efeitos dos fármacos , Streptomyces griseus/crescimento & desenvolvimento , Toxinas Biológicas/biossíntese
13.
Nat Commun ; 12(1): 1352, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649340

RESUMO

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins.


Assuntos
Centrossomo/metabolismo , Polirribossomos/metabolismo , Transporte de RNA , Animais , Proteínas de Ciclo Celular/metabolismo , Centrossomo/efeitos dos fármacos , Cicloeximida/farmacologia , Drosophila/genética , Células HeLa , Humanos , Mitose/efeitos dos fármacos , Fases de Leitura Aberta/genética , Polirribossomos/efeitos dos fármacos , Puromicina/farmacologia , Transporte de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo
14.
Life Sci Alliance ; 4(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33536237

RESUMO

γ-secretase inhibitors (GSI) were developed to reduce the generation of Aß peptide to find new Alzheimer's disease treatments. Clinical trials on Alzheimer's disease patients, however, showed several side effects that worsened the cognitive symptoms of the treated patients. The observed side effects were partially attributed to Notch signaling. However, the effect on other γ-secretase substrates, such as the p75 neurotrophin receptor (p75NTR) has not been studied in detail. p75NTR is highly expressed in the basal forebrain cholinergic neurons (BFCNs) during all life. Here, we show that GSI treatment induces the oligomerization of p75CTF leading to the cell death of BFCNs, and that this event is dependent on TrkA activity. The oligomerization of p75CTF requires an intact cholesterol recognition sequence (CRAC) and the constitutive binding of TRAF6, which activates the JNK and p38 pathways. Remarkably, TrkA rescues from cell death by a mechanism involving the endocytosis of p75CTF. These results suggest that the inhibition of γ-secretase activity in aged patients, where the expression of TrkA in the BFCNs is already reduced, could accelerate cholinergic dysfunction and promote neurodegeneration.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Endocitose , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Morte Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica/efeitos dos fármacos , Proteólise , Receptor de Fator de Crescimento Neural/química
15.
Development ; 148(5)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33593817

RESUMO

The shoot apical meristem (SAM) is a reservoir of stem cells that gives rise to all post-embryonic above-ground plant organs. The size of the SAM remains stable over time owing to a precise balance of stem cell replenishment versus cell incorporation into organ primordia. The WUSCHEL (WUS)/CLAVATA (CLV) negative feedback loop is central to SAM size regulation. Its correct function depends on accurate spatial expression of WUS and CLV3 A signaling pathway, consisting of ERECTA family (ERf) receptors and EPIDERMAL PATTERNING FACTOR LIKE (EPFL) ligands, restricts SAM width and promotes leaf initiation. Although ERf receptors are expressed throughout the SAM, EPFL ligands are expressed in its periphery. Our genetic analysis of Arabidopsis demonstrated that ERfs and CLV3 synergistically regulate the size of the SAM, and wus is epistatic to ERf genes. Furthermore, activation of ERf signaling with exogenous EPFLs resulted in a rapid decrease of CLV3 and WUS expression. ERf-EPFL signaling inhibits expression of WUS and CLV3 in the periphery of the SAM, confining them to the center. These findings establish the molecular mechanism for stem cell positioning along the radial axis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Homeodomínio/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/farmacologia , Cicloeximida/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Meristema/fisiologia , Mutagênese , Folhas de Planta/metabolismo
16.
Cancer Res ; 81(8): 2002-2014, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33632898

RESUMO

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5' untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC. SIGNIFICANCE: These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.


Assuntos
Adenocarcinoma/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Quadruplex G , Genes ras/genética , Humanos , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Oxirredução , Neoplasias Pancreáticas/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Helicases , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Triterpenos/farmacologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo
17.
Biochim Biophys Acta Mol Cell Res ; 1868(6): 118968, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33454316

RESUMO

Retinoic acid (RA) induces granulocytic differentiation and inhibits the growth of human promyelocytic leukemia HL60 cells. α-Actinin-4 is a member of the α-actinin family, which exhibits unique mechanosensory regulation. Herein, we elucidated the effects of RA on α-actinin-4 expression during cell differentiation. RA increased the levels of α-actinin-4 protein significantly, while mRNA expression remained unchanged. In addition, RA treatment altered the intracellular localization of α-actinin-4 from the nucleus to the cytoplasm. Cells pretreated with RA, maintained α-actinin-4 protein levels after cycloheximide treatment as compared with control cells. The amount of ubiquitylated α-actinin-4 protein in RA-treated cells was less than in control cells. These results indicate that RA may inhibit nuclei transport and proteasomal degradation of α-actinin-4 protein. α-Actinin-4 may play a significant role in RA-induced differentiation, including the promotion of cytomorphology changes.


Assuntos
Actinina/metabolismo , Cicloeximida/farmacologia , Leucemia Promielocítica Aguda/metabolismo , Tretinoína/farmacologia , Regulação para Cima , Actinina/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Proteólise , Ubiquitinação
18.
J Asian Nat Prod Res ; 23(2): 110-116, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31885279

RESUMO

A couple of new cycloheximide epimers, 13(α)-acetoxy-anhydroisoheximide (1) and 13(ß)-acetoxy-anhydroisoheximide (2), together with six known compounds (3-8), were obtained from the cultures of Streptomyces sp. YG7. The structures were elucidated based on a comprehensive spectroscopic data analysis including 1D and 2D NMR, as well as HRESIMS, and by comparison with the literature. The X-ray crystal analysis of 1 further confirmed the structure. All the compounds were tested for antifungal activity. Compounds 1, 2 and 5-8 showed moderate Canidia albicans inhibitory activity, while 5 and 6 presented moderate Pyricularia oryzae inhibitory activity. [Formula: see text].


Assuntos
Streptomyces , Antifúngicos/farmacologia , Ascomicetos , Cicloeximida , Estrutura Molecular
19.
FEBS J ; 288(1): 142-159, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32543048

RESUMO

Charcot-Marie-Tooth disease (CMT) encompasses a set of genetically and clinically heterogeneous neuropathies characterized by length-dependent dysfunction of the peripheral nervous system. Mutations in over 80 diverse genes are associated with CMT, and aminoacyl-tRNA synthetases (ARS) constitute a large gene family implicated in the disease. Despite considerable efforts to elucidate the mechanistic link between ARS mutations and the CMT phenotype, the molecular basis of the pathology is unknown. In this work, we investigated the impact of three CMT-associated substitutions (V155G, Y330C, and R137Q) in the cytoplasmic histidyl-tRNA synthetase (HARS1) on neurite outgrowth and peripheral nervous system development. The model systems for this work included a nerve growth factor-stimulated neurite outgrowth model in rat pheochromocytoma cells (PC12), and a zebrafish line with GFP/red fluorescent protein reporters of sensory and motor neuron development. The expression of CMT-HARS1 mutations led to attenuation of protein synthesis and increased phosphorylation of eIF2α in PC12 cells and was accompanied by impaired neurite and axon outgrowth in both models. Notably, these effects were phenocopied by histidinol, a HARS1 inhibitor, and cycloheximide, a protein synthesis inhibitor. The mutant proteins also formed heterodimers with wild-type HARS1, raising the possibility that CMT-HARS1 mutations cause disease through a dominant-negative mechanism. Overall, these findings support the hypothesis that CMT-HARS1 alleles exert their toxic effect in a neuronal context, and lead to dysregulated protein synthesis. These studies demonstrate the value of zebrafish as a model for studying mutant alleles associated with CMT, and for characterizing the processes that lead to peripheral nervous system dysfunction.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Histidina-tRNA Ligase/genética , Crescimento Neuronal/genética , Neurônios/metabolismo , Sistema Nervoso Periférico/metabolismo , Biossíntese de Proteínas , Animais , Animais Geneticamente Modificados , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Cicloeximida/farmacologia , Modelos Animais de Doenças , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina-tRNA Ligase/antagonistas & inibidores , Histidina-tRNA Ligase/metabolismo , Histidinol/farmacologia , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Células PC12 , Sistema Nervoso Periférico/patologia , Multimerização Proteica , Ratos , Peixe-Zebra
20.
J Mol Neurosci ; 71(1): 169-177, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32602030

RESUMO

Traumatic brain injury (TBI) is one of the leading causes of death worldwide. Long non-coding RNAs (LncRNAs) have been reported to be closely associated with various diseases, but their roles in TBI has not been fully elucidated. The purpose of this study was to elucidate the underlying mechanism of LncRNA HOTAIR in TBI-induced microglial activation and inflammatory factor release. In vivo mouse TBI model and in vitro microglia activation model were established by Feeney's free-fall impact method and by LPS stimulation, respectively. The expression of LncRNA HOTAIR in activated microglia was detected by qRT-PCR. After shRNA knocked down, the expressions of LncRNA HOTAIR and microglia activation marker Iba-1 in microglia were detected by qRT-PCR and Western blot and by ELISA that detected the concentration of inflammatory factor in cell culture supernatants. The relationship between LncRNA HOTAIR and MYD88 in mouse microglia BV2 cells was observed by RNA pull-down assay. Furthermore, the effect of LncRNA HOTAIR on MYD88 stability was assessed by cycloheximide (CHX)-chase and by immunoprecipitation and ubiquitination assays that analyzed MYD88 ubiquitination. LncRNA HOTAIR was abnormally highly expressed in activated microglia. By Western blot and ELISA, the knockdown of LncRNA HOTAIR in microglia significantly repressed microglia activation and inflammatory factor release. By RNA pull-down assay, LncRNA HOTAIR could bind to MYD88 protein. Besides, by cycloheximide (CHX)-chase and immunoprecipitation and ubiquitination assays, the overexpression of the LncRNA HOTAIR enhanced the stability of MYD88 protein and inhibited Nrdp1-mediated ubiquitination of MYD88 protein. After the transfection of shRNA-HOTAIR and shRNA-HOTAIR+pcDNA-MYD88 into microglia, shRNA-HOTAIR could significantly inhibit the activation of microglia and the release of inflammatory factors, while these effects were reversed after the transfection of pcDNA-MYD88. Our experimental data indicated that LncRNA HOTAIR was highly expressed in activated microglia, and our further studies had found that the interference with LncRNA HOTAIR could repress microglia activation and inflammatory factor release via promoting Nrdp1-mediated ubiquitination of MYD88 protein.


Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Microglia/fisiologia , Fator 88 de Diferenciação Mieloide/metabolismo , Processamento de Proteína Pós-Traducional , RNA Longo não Codificante/genética , Animais , Células Cultivadas , Córtex Cerebral/citologia , Cicloeximida/farmacologia , Inflamação , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/lesões , Interferência de RNA , RNA Interferente Pequeno/genética , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação
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