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1.
EMBO J ; 39(17): e105696, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32716134

RESUMEN

Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.


Asunto(s)
Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Núcleo Celular/genética , Retículo Endoplásmico/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de la Membrana/genética , Ratones , Oryzias
2.
Stem Cell Reports ; 16(5): 1381-1390, 2021 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-33891873

RESUMEN

Controlling cell fate has great potential for regenerative medicine, drug discovery, and basic research. Although transcription factors are able to promote cell reprogramming and transdifferentiation, methods based on their upregulation often show low efficiency. Small molecules that can facilitate conversion between cell types can ameliorate this problem working through safe, rapid, and reversible mechanisms. Here, we present DECCODE, an unbiased computational method for identification of such molecules based on transcriptional data. DECCODE matches a large collection of drug-induced profiles for drug treatments against a large dataset of primary cell transcriptional profiles to identify drugs that either alone or in combination enhance cell reprogramming and cell conversion. Extensive validation in the context of human induced pluripotent stem cells shows that DECCODE is able to prioritize drugs and drug combinations enhancing cell reprogramming. We also provide predictions for cell conversion with single drugs and drug combinations for 145 different cell types.


Asunto(s)
Reprogramación Celular , Bibliotecas de Moléculas Pequeñas/farmacología , Algoritmos , Animales , Automatización , Reprogramación Celular/efectos de los fármacos , Análisis por Conglomerados , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Reproducibilidad de los Resultados
3.
Hum Gene Ther ; 29(8): 886-901, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29641320

RESUMEN

Retinal gene therapy based on adeno-associated viral (AAV) vectors is safe and efficient in humans. The low intrinsic DNA transfer capacity of AAV has been expanded by dual vectors where a large expression cassette is split in two halves independently packaged in two AAV vectors. Dual AAV transduction efficiency, however, is greatly reduced compared to that obtained with a single vector. As AAV intracellular trafficking and processing are negatively affected by phosphorylation, this study set to identify kinase inhibitors that can increase dual AAV vector transduction. By high-throughput screening of a kinase inhibitors library, three compounds were identified that increase AAV transduction in vitro, one of which has a higher effect on dual than on single AAV vectors. Importantly, the transduction enhancement is exerted on various AAV serotypes and is not transgene dependent. As kinase inhibitors are promiscuous, siRNA-mediated silencing of targeted kinases was performed, and AURKA and B, PLK1, and PTK2 were among those involved in the increase of AAV transduction levels. The study shows that kinase inhibitor administration reduces AAV serotype 2 (AAV2) capsid phosphorylation and increases the activity of DNA-repair pathways involved in AAV DNA processing. Importantly, the kinase inhibitor PF-00562271 improves dual AAV8 transduction in photoreceptors following sub-retinal delivery in mice. The study identifies kinase inhibitors that increase dual and single AAV transduction by modulating AAV entry and post-entry steps.


Asunto(s)
Terapia Genética , Vectores Genéticos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Retina/metabolismo , Transducción Genética , Animales , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Dependovirus/genética , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/genética , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/uso terapéutico , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/virología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Retina/patología , Retina/virología , Quinasa Tipo Polo 1
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