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1.
Methods Mol Biol ; 2320: 247-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302663

RESUMO

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Introdução de Genes/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Células Cultivadas , Células Clonais , Meios de Cultura , Primers do DNA , Resistência a Medicamentos/genética , Eletroporação , Células-Tronco Embrionárias/citologia , Edição de Genes/métodos , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Puromicina/farmacologia , RNA Guia/genética , Reparo de DNA por Recombinação/genética
2.
Methods Mol Biol ; 2320: 235-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302662

RESUMO

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) are powerful tools for elucidating the pathology behind inherited cardiomyopathies. Genome editing technologies enable targeted genome replacement and the generation of isogenic hiPSCs, allowing investigators to precisely determine the roles of identified mutations. Here, we describe a protocol to obtain isogenic hiPSCs with the corrected allele via homology-directed repair (HDR) using CRISPR/Cas9 genome editing under feeder-free conditions. Seeding hiPSCs in a 24-well plate and conducting the initial evaluation using direct genomic sequencing after 1 week is cost- and time-effective. Following optimization of the protocol, sequence confirmation of the corrected HDR clone is completed within 21 days.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Diferenciação Celular , Células Clonais/citologia , Células Clonais/metabolismo , Eletroporação/métodos , Desenho de Equipamento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Puromicina/farmacologia , Reparo de DNA por Recombinação
3.
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
4.
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
5.
RNA ; 27(4): 411-419, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33479117

RESUMO

Ribosomes are the macromolecular machines at the heart of protein synthesis; however, their function can be modulated by a variety of additional protein factors that directly interact with them. Here, we report the cryo-EM structure of human Ebp1 (p48 isoform) bound to the human 80S ribosome at 3.3 Å resolution. Ebp1 binds in the vicinity of the peptide exit tunnel on the 80S ribosome, and this binding is enhanced upon puromycin-mediated translational inhibition. The association of Ebp1 with the 80S ribosome centers around its interaction with ribosomal proteins eL19 and uL23 and the 28S rRNA. Further analysis of the Ebp1-ribosome complex suggests that Ebp1 can rotate around its insert domain, which may enable it to assume a wide range of conformations while maintaining its interaction with the ribosome. Structurally, Ebp1 shares homology with the methionine aminopeptidase 2 family of enzymes; therefore, this inherent flexibility may also be conserved.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Biossíntese de Proteínas , RNA Ribossômico/química , Proteínas de Ligação a RNA/química , Proteínas Ribossômicas/química , Ribossomos/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Termodinâmica
6.
Endocrinology ; 162(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33367617

RESUMO

A correct biosynthetic activity is thought to be essential for the long-term function and survival of islet cells in culture and possibly also after islet transplantation. Compared to the secretory activity, biosynthetic activity has been poorly studied in pancreatic islet cells. Here we aimed to assess biosynthetic activity at the single cell level to investigate if protein synthesis is dependent on secretagogues and increased as a consequence of hormonal secretion. Biosynthetic activity in rat islet cells was studied at the single cell level using O-propargyl-puromycin (OPP) that incorporates into newly translated proteins and chemically ligates to a fluorescent dye by "click" reaction. Heterogeneous biosynthetic activity was observed between the four islet cell types, with delta cells showing the higher relative protein biosynthesis. Beta cells protein biosynthesis was increased in response to glucose while 3-isobutyl-1-methylxanthine and phorbol-12-myristate-13-acetate, 2 drugs known to stimulate insulin secretion, had no similar effect on protein biosynthesis. However, after several hours of secretion, protein biosynthesis remained high even when cells were challenged to basal conditions. These results suggest that mechanisms regulating secretion and biosynthesis in islet cells are different, with glucose directly triggering beta cells protein biosynthesis, independently of insulin secretion. Furthermore, this OPP labeling approach is a promising method to identify newly synthesized proteins under various physiological and pathological conditions.


Assuntos
Glucose/farmacologia , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Células Cultivadas , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Puromicina/análogos & derivados , Puromicina/farmacologia , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem
7.
Nucleic Acids Res ; 49(1): 547-567, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330920

RESUMO

Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.


Assuntos
Bacteroidetes/genética , Iniciação Traducional da Cadeia Peptídica , Sequências Reguladoras de Ácido Ribonucleico , Ribossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Códon de Iniciação , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Flavobacterium/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Puromicina/farmacologia , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , RNA Ribossômico 5S/genética , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência , Especificidade da Espécie
8.
PLoS One ; 15(12): e0242426, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33284815

RESUMO

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500µm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.


Assuntos
Células Amácrinas/fisiologia , Cultura Primária de Células/métodos , Células Ganglionares da Retina/fisiologia , Células Amácrinas/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Concentração Inibidora 50 , Masculino , Camundongos , Crescimento Neuronal , Puromicina/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos
9.
Elife ; 92020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33252038

RESUMO

Wnt signaling is downregulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way toward novel therapeutic modalities. We demonstrate that EHMT2 suppresses canonical Wnt signaling by activating expression of the Wnt antagonist DKK1. Inhibition of EHMT2 expression or activity in human ERMS cell lines reduced DKK1 expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in mouse xenograft models in vivo. Mechanistically, EHMT2 impacted Sp1 and p300 enrichment at the DKK1 promoter. The reduced tumor growth upon EHMT2 deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among 13 drugs targeting chromatin modifiers, EHMT2 inhibitors were highly effective in reducing ERMS cell viability. Our study demonstrates that ERMS cells are vulnerable to EHMT2 inhibitors and suggest that targeting the EHMT2-DKK1-ß-catenin node holds promise for differentiation therapy.


Assuntos
Epigênese Genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Rabdomiossarcoma Embrionário/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Dimetil Sulfóxido/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Camundongos Nus , Puromicina/farmacologia , Pirazinas/farmacologia , Piridinas/farmacologia , Quinazolinas/farmacologia , Interferência de RNA , Rabdomiossarcoma Embrionário/genética
10.
PLoS One ; 15(10): e0240480, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33079945

RESUMO

Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted treatments are necessary to manage the disease. Currently, no tools for genetic manipulation are available to identify and test specific drug targets in these fungi. To facilitate the development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. We have identified effective concentrations of each for selection in both liquid culture and on solid media. These concentrations are within the range of concentrations used for selecting genetically modified cells from a variety of other eukaryotic species.


Assuntos
Anfíbios/microbiologia , Antifúngicos/farmacologia , Batrachochytrium/efeitos dos fármacos , Batrachochytrium/crescimento & desenvolvimento , Micologia/métodos , Animais , Batrachochytrium/genética , Bleomicina/farmacologia , Cinamatos/farmacologia , Testes Diagnósticos de Rotina , Avaliação Pré-Clínica de Medicamentos , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Testes de Sensibilidade Microbiana , Neomicina/farmacologia , Puromicina/farmacologia , Pirrolidinonas/farmacologia , Seleção Genética
11.
Int J Biol Macromol ; 165(Pt A): 1373-1381, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33045297

RESUMO

Puromycin sensitive aminopeptidase (PSA or NPEPPS) is a M1 class aminopeptidase is selectively inhibited by the natural product puromycin, an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. The molecular basis for this selective inhibition has not been understood well. Here, we report the basis for selectivity of puromycin using biochemical, structural and molecular modeling tools on four different M1 family enzymes including human PSA. Except for PSA, the other three enzymes were not inhibited. Instead, the peptide bond in the puromycin is hydrolyzed to O-methyl-L-tyrosine (OMT) and puromycin aminonucleoside (PAN). Neither of the hydrolyzed products, individually or together inhibit any of the four enzymes. Crystal structure of ePepN using crystals that are incubated with puromycin contained the hydrolyzed products instead of intact puromycin. On the other hand, intact puromycin molecule was observed in the crystal structure of the inactive mutant ePepN (E298A)-puromycin complex. Surprisingly, puromycin does not enter the active site of the mutant enzyme but binds near the entrance. Comparison of puromycin binding region in ePepN mutant enzyme and molecular modeling studies suggest that PSA might be inhibited by similar mode of binding there by blocking the entrance of the active site.


Assuntos
Modelos Moleculares , Antígeno Prostático Específico/antagonistas & inibidores , Conformação Proteica , Puromicina/química , Sequência de Aminoácidos/genética , Escherichia coli/genética , Humanos , Cinética , Masculino , Antígeno Prostático Específico/química , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/ultraestrutura , Puromicina/farmacologia , Especificidade por Substrato/genética
12.
Methods Mol Biol ; 2203: 231-238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833216

RESUMO

Ribopuromycylation enables the visualization and quantitation of translation on a cellular level by immunofluorescence or in total using standard western blotting. This technique uses ribosome catalyzed puromycylation of nascent chains followed by immobilization on the ribosome by antibiotic chain elongation inhibitor emetine. Detection of puromycylated ribosome-bound nascent chains can then be achieved using a puromycin-specific antibody.


Assuntos
Coronavirus/genética , Puromicina/farmacologia , Infecções por Coronavirus , Imunofluorescência , Interações Hospedeiro-Patógeno , Humanos , Biossíntese de Proteínas , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
13.
Elife ; 92020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32844746

RESUMO

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.


Assuntos
Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Inibidores da Síntese de Proteínas , Puromicina , Ribossomos , Animais , Linhagem Celular Tumoral , Camundongos , Inibidores da Síntese de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas/química , Proteínas/metabolismo , Puromicina/metabolismo , Puromicina/farmacologia , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
14.
Elife ; 92020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32844748

RESUMO

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.


Assuntos
Núcleo Celular , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas , Puromicina , Animais , Caenorhabditis elegans , Núcleo Celular/química , Núcleo Celular/metabolismo , Emetina/metabolismo , Emetina/farmacologia , Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Inibidores da Síntese de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/metabolismo , Puromicina/farmacologia , RNA de Transferência/metabolismo , Coelhos , Ribossomos/metabolismo , Análise de Célula Única
15.
Biochem Biophys Res Commun ; 531(2): 112-117, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32782144

RESUMO

Iron deficiency anemia indicates poor nutrition and is a public health problem. Iron deficiency is also associated with muscle weakness. However, the intracellular mechanisms by which iron deficiency induces muscle weakness are obscure. The purpose of the present study was to evaluate the effect of iron deficiency on protein synthesis in basal and branched-amino acids (BCAA)- and insulin-stimulated state in muscle cells. Differentiated C2C12 myotubes were incubated with an iron chelator, deferoxamine mesylate, and then stimulated with BCAA or insulin to activate protein synthesis. This iron deprivation resulted in a significant reduction in the abundance of iron-containing proteins, such as the mitochondrial complex 1 subunit protein, compared to control cells, but not of protein that does not contain iron, such as citrate synthase. Proteins involved in glucose utilization, such as glucose transpoter-1, hexokinase and AMP-activated protein kinase (AMPK), were upregulated under iron deficiency. Additionally, rates of BCAA- and insulin-stimulated protein synthesis, measured by puromycin incorporation, were lower in iron-deficient myotubes than in control cells. We suggest that low iron availability attenuates BCAA- and insulin-stimulated protein synthesis, possibly via activation of AMPK in myotubes. The present findings advance the understanding of the importance of iron to skeletal muscle protein synthesis and, thus, may contribute to the prevention of sarcopenia and frailty.


Assuntos
Aminoácidos de Cadeia Ramificada/farmacologia , Insulina/farmacologia , Ferro/deficiência , Fibras Musculares Esqueléticas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipase/genética , Lipase/metabolismo , Camundongos , Proteínas Mitocondriais/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina
16.
Nat Commun ; 11(1): 2106, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355156

RESUMO

Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The system exploits the toxicity of commonly used broad-spectrum antibiotics geneticin and puromycin to kill the non-rescued sex. Sex-specific rescue is achieved by inserting sex-specific introns into the coding sequences of antibiotic-resistance genes. When raised on geneticin-supplemented food, the sex-sorter line establishes 100% positive selection for female progeny, while the food supplemented with puromycin positively selects 100% male progeny. Since the described system exploits conserved sex-specific splicing mechanisms and reagents, it has the potential to be adaptable to other insect species of medical and agricultural importance.


Assuntos
Drosophila melanogaster/efeitos dos fármacos , Engenharia Genética/métodos , Gentamicinas/farmacologia , Puromicina/farmacologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Resistência a Medicamentos , Éxons , Feminino , Genética Populacional , Homozigoto , Íntrons , Masculino , Controle de Pragas , Splicing de RNA , Análise para Determinação do Sexo
17.
Cells ; 9(5)2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32466308

RESUMO

During myocardial infarction, dysregulation of Ca2+ homeostasis between the reticulum, mitochondria, and cytosol occurs in cardiomyocytes and leads to cell death. Ca2+ leak channels are thought to be key regulators of the reticular Ca2+ homeostasis and cell survival. The present study aimed to determine whether a particular reticular Ca2+ leak channel, the translocon, also known as translocation channel, could be a relevant target against ischemia/reperfusion-mediated heart injury. To achieve this objective, we first used an intramyocardial adenoviral strategy to express biosensors in order to assess Ca2+ variations in freshly isolated adult mouse cardiomyocytes to show that translocon is a functional reticular Ca2+ leak channel. Interestingly, translocon activation by puromycin mobilized a ryanodine receptor (RyR)-independent reticular Ca2+ pool and did not affect the excitation-concentration coupling. Second, puromycin pretreatment decreased mitochondrial Ca2+ content and slowed down the mitochondrial permeability transition pore (mPTP) opening and the rate of cytosolic Ca2+ increase during hypoxia. Finally, this translocon pre-activation also protected cardiomyocytes after in vitro hypoxia reoxygenation and reduced infarct size in mice submitted to in vivo ischemia-reperfusion. Altogether, our report emphasizes the role of translocon in cardioprotection and highlights a new paradigm in cardioprotection by functionally uncoupling the RyR-dependent Ca2+ stores and translocon-dependent Ca2+ stores.


Assuntos
Cálcio/metabolismo , Cardiotônicos/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Canais de Translocação SEC/metabolismo , Animais , Acoplamento Excitação-Contração , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Puromicina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
18.
Clin Exp Immunol ; 201(2): 205-221, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32403163

RESUMO

Regulatory T (Treg ) cell therapy is a promising approach for immune tolerance induction in autoimmunity conditions and cell/organ transplantations. Insufficient isolation yields and impurity during downstream processes and Treg instability after adoptive transfer in inflammatory conditions are major limitations to Treg therapy, and indicate the importance of seeking a valid, reliable method for de-novo generation of Tregs . In this research, we evaluated Treg -like cells obtained from different Treg differentiation protocols in terms of their yield, purity and activity. Differentiation was performed on naive CD4+ cells and a naive CD4+ /Treg co-culture by using three different protocols - ectopic expression of forkhead box protein P3 (E-FoxP3), soluble transforming growth factor ß (S-TGF) and small molecules [N-acetyl puromycin and SR1555 (N-Ac/SR)]. The results showed that a high yield of a homogeneous population of Treg -like cells could be achieved by the N-Ac/SR method under a T helper type 17 (Th17)-polarizing condition, particularly interleukin (IL)-6 and TGF-ß, when compared with the E-FoxP3 and S-TGF methods. Surprisingly, SR completely inhibited the differentiation of IL-17-producing cells and facilitated Treg generation in the inflammatory condition and had highly suppressive activity against T cell proliferation without Treg -specific demethylase region (TSDR) demethylation. For the first time, to our knowledge, we report the generation of efficient, pure Treg -like cells by using small molecules during in-vitro inflammatory conditions. Our results suggested that the N-Ac/SR method has several advantages for Treg generation when compared with the other methods, including a higher purity of Tregs , easier procedure, superior suppressive activity during the inflammatory condition and decreased cost.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Transferência Adotiva , Compostos de Bifenilo/farmacologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Humanos , Inflamação , Interleucina-2/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Piperazinas/farmacologia , Puromicina/farmacologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
19.
Cells ; 9(1)2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936702

RESUMO

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


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
20.
Autophagy ; 16(2): 334-346, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31177902

RESUMO

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.


Assuntos
Autofagia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Streptococcus pyogenes/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Células HeLa , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , NAD+ Nucleosidase/metabolismo , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Puromicina/farmacologia , Estreptolisinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
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