Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros

Base de dados
Tipo de documento
País de afiliação
Intervalo de ano de publicação
1.
Cell ; 155(4): 909-921, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-24209627

RESUMO

Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3ß inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3ß inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Desenvolvimento Muscular/efeitos dos fármacos , Animais , Colforsina/farmacologia , Técnicas de Cultura , AMP Cíclico/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Distrofias Musculares/terapia , Células Satélites de Músculo Esquelético/metabolismo , Transplante de Células-Tronco , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
2.
J Am Chem Soc ; 137(24): 7929-34, 2015 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-26042473

RESUMO

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic ß-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote ß-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat ß cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/imunologia , Janus Quinase 2/imunologia , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Fator de Transcrição STAT1/imunologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/imunologia , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina Tiolesterase/imunologia , Ubiquitinação/efeitos dos fármacos
3.
Science ; 368(6486): 78-84, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32241946

RESUMO

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo-designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.


Assuntos
Receptor Celular 2 do Vírus da Hepatite A/química , Engenharia de Proteínas , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Receptor Celular 2 do Vírus da Hepatite A/genética , Humanos , Lógica , Espectrometria de Massas , Biologia Sintética , Linfócitos T/metabolismo , Transcrição Gênica , Leveduras/metabolismo
4.
Cell Chem Biol ; 25(12): 1506-1518.e13, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30318461

RESUMO

Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.


Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Tetraciclinas/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , RNA Ribossômico/genética , Relação Estrutura-Atividade , Tetraciclinas/química
5.
Elife ; 62017 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-28832322

RESUMO

The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Peixe-Zebra/embriologia , Animais , Ácidos Cafeicos/metabolismo , Crista Neural/embriologia , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo
6.
Science ; 351(6272): aad2197, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26823433

RESUMO

The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.


Assuntos
Carcinogênese/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/genética , Melanoma/genética , Crista Neural/metabolismo , Neoplasias Cutâneas/genética , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Melanócitos/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de Transcrição SOXE/genética , Proteína Supressora de Tumor p53/genética , Proteínas de Peixe-Zebra/genética
7.
Science ; 343(6168): 301-5, 2014 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-24292625

RESUMO

Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.


Assuntos
Antineoplásicos/farmacologia , Fator de Transcrição Ikaros/metabolismo , Mieloma Múltiplo/metabolismo , Talidomida/análogos & derivados , Linhagem Celular Tumoral , Células HEK293 , Humanos , Fator de Transcrição Ikaros/genética , Interleucina-2/biossíntese , Lenalidomida , Proteólise , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Talidomida/farmacologia , Ubiquitinação
8.
Cancer Discov ; 3(4): 382-3, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23580282

RESUMO

In this issue of Cancer Discovery, Pedersen and colleagues present the first mouse model of primary CNS melanoma, which arises when oncogenic NRAS is expressed from the endogenous Nras promoter in melanocytes during embryogenesis. In support of this model, two pediatric cases of NRAS-mutant primary melanoma of the CNS are identified.


Assuntos
Neoplasias do Sistema Nervoso Central/genética , Genes ras/genética , Melanócitos/metabolismo , Melanoma/genética , Animais , Feminino , Humanos , Masculino
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA