RESUMO
Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice.
Assuntos
Corantes Fluorescentes/análise , Proteínas Luminescentes/análise , Imagem Óptica/métodos , Animais , Linhagem Celular Tumoral , Drosophila/ultraestrutura , Feminino , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Neoplasias/diagnóstico , Fotodegradação , Conformação Proteica , Estabilidade Proteica , Peixe-Zebra/embriologia , Proteína Vermelha FluorescenteRESUMO
MYC proteins are major drivers of cancer yet are considered undruggable because their DNA binding domains are composed of two extended alpha helices with no apparent surfaces for small-molecule binding. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora A. We describe a class of inhibitors that disrupts the native conformation of Aurora A and drives the degradation of MYCN protein across MYCN-driven cancers. Comparison of cocrystal structures with structure-activity relationships across multiple inhibitors and chemotypes, coupled with mechanistic studies and biochemical assays, delineates an Aurora A conformation-specific effect on proteolytic degradation of MYCN, rather than simple nanomolar-level inhibition of Aurora A kinase activity.
Assuntos
Antineoplásicos/farmacologia , Aurora Quinase A/química , Neuroblastoma/tratamento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Regulação Alostérica , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Área Sob a Curva , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Modelos Moleculares , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/patologia , Proteínas Nucleares/química , Proteínas Oncogênicas/química , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacocinética , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteólise , Pirimidinas/química , Pirimidinas/farmacocinética , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Relação Estrutura-Atividade , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging, and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the haeme oxygenase responsible for BV biosynthesis and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared with monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labelling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumours in whole mice. Our work shows promise in the application of IFPs for protein labelling and in vivo imaging.
Assuntos
Neoplasias Encefálicas/diagnóstico , Corantes Fluorescentes/metabolismo , Proteínas Luminescentes/metabolismo , Neuroimagem/métodos , Neurônios/metabolismo , Animais , Biliverdina/metabolismo , Neoplasias Encefálicas/metabolismo , Cristalografia por Raios X , Drosophila , Células HEK293 , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Raios Infravermelhos , Larva , Camundongos , Microscopia Confocal , Fitocromo , RatosRESUMO
Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.
Assuntos
Neuroblastoma/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Azepinas/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Criança , Regulação para Baixo/efeitos dos fármacos , Feminino , Amplificação de Genes , Humanos , Camundongos , Terapia de Alvo Molecular , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Nucleares/deficiência , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transfecção , Triazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma, a tumor of the developing peripheral sympathetic nervous system, is the most common and deadly extracranial solid tumor of childhood. Risk-stratification and risk-adapted therapy play a large role in the modern treatment of neuroblastoma. Recently, through extensive international collaboration, new guidelines for risk stratification have emerged that will aid in international cooperative studies, as well as clarifying therapeutic options for patients. Current therapies for low- and intermediate-risk neuroblastoma have resulted in excellent prognoses for these risk strata, and current efforts are concentrated on chemotherapy reduction. By contrast, much more gradual progress has been made in improving survival for high-risk neuroblastoma patients, despite significant chemotherapy intensification. Current investigations focus on overcoming resistance by elucidating the molecular/genetic causes of neuroblastoma tumorigenesis and progression, with the aim of developing more effective biologically targeted therapies for this disease.