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1.
Mol Cell ; 66(4): 517-532.e9, 2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28525743

RESUMO

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.


Assuntos
Autofagia , Carcinoma Ductal Pancreático/metabolismo , Lisossomos/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/genética , Cromatina/metabolismo , Regulação para Baixo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Agregados Proteicos , Ligação Proteica , Proteólise , Interferência de RNA , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Transfecção
2.
Int J Mol Sci ; 20(9)2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-31060328

RESUMO

The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens. Arf6 is also used in the signaling of Toll-like receptors and in the activation of NADPH oxidases. In this review, we first give an overview of the different roles and mechanisms of action of Arf6 and then focus on its role in innate immunity and host-pathogen interactions.


Assuntos
Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Fator 6 de Ribosilação do ADP , Animais , Biomarcadores , Ativação Enzimática , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Fagocitose/genética , Fagocitose/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Virulência
3.
J Biol Chem ; 289(3): 1364-76, 2014 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-24297182

RESUMO

Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-κB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal(-/-) mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-dependent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/Trif-dependent TLR4 signaling.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Macrófagos/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Animais , Linhagem Celular , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Camundongos , Camundongos Knockout , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores de Interleucina/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia
4.
J Biol Chem ; 288(17): 12054-66, 2013 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-23460645

RESUMO

Upon activation, Toll-like receptor 4 (TLR4) binds adapter proteins, including MyD88 (myeloid differentiation primary response gene 88) and Mal (MyD88 adapter-like) for its signal transduction. TLR4 and the adapter proteins each contain a Toll/Il-1 receptor domain (TIR domain). In this study we used random mutagenesis and the mammalian two-hybrid method MAPPIT (mammalian protein-protein interaction trap) to identify mutations in Mal that disrupt its interaction with TLR4 and/or MyD88. Our study shows that four potential binding sites and the AB-loop in the Mal TIR domain all contribute to formation of the TLR4-Mal-MyD88 complex. Mutations in the symmetrical back-to-back Mal homodimer interface affect Mal homodimerization and interaction with MyD88 and TLR4. Our data suggest that Mal dimerization may lead to formation of potential binding platforms on the top and the side of the Mal dimer that bind MyD88 or TLR4. Mutations that affect the interaction of Mal with MyD88 also affect NF-κB activation induced by Mal overexpression. In MAPPIT, co-expression of the MyD88 TIR domain enhances Mal dimerization and Mal binding to TLR4. Similarly, co-expression of Mal and the MyD88 TIR domain strongly promotes dimerization of the TLR4 intracellular domain in MAPPIT. The different types of TIR-TIR interactions in the TLR4-Mal-MyD88 complex thus show cooperative binding in MAPPIT. We present plausible models for the TIR-TIR interactions in the TLR4-Mal-MyD88 complex.


Assuntos
Sítios de Ligação , Modelos Biológicos , Fator 88 de Diferenciação Mieloide , Multimerização Proteica/fisiologia , Receptor 4 Toll-Like , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Mapeamento de Peptídeos/métodos , Ligação Proteica , Receptores de Interleucina-1/química , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
5.
Methods Mol Biol ; 1880: 359-374, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610710

RESUMO

Autophagy is a highly regulated process, and its deregulation can contribute to various diseases, including cancer, immune diseases, and neurodegenerative disorders. Here we describe the design, protocol, and analysis of an imaging-based high-throughput screen with an endogenous autophagy readout. The screen uses a genome-wide siRNA library to identify autophagy regulators in mammalian cells.


Assuntos
Autofagia/genética , Técnicas de Silenciamento de Genes/métodos , RNA Interferente Pequeno/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular , Técnicas de Silenciamento de Genes/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Proteínas Associadas aos Microtúbulos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transfecção/instrumentação , Transfecção/métodos
6.
Cancer Res ; 79(8): 1884-1898, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30765601

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is driven by metabolic changes in pancreatic cells caused by oncogenic mutations and dysregulation of p53. PDAC cell lines and PDAC-derived xenografts grow as a result of altered metabolic pathways, changes in stroma, and autophagy. Selective targeting and inhibition of one of these may open avenues for the development of new therapeutic strategies. In this study, we performed a genome-wide siRNA screen in a PDAC cell line using endogenous autophagy as a readout and identified several regulators of autophagy that were required for autophagy-dependent PDAC cell survival. Validation of two promising candidates, MPP7 (MAGUK p55 subfamily member 7, a scaffolding protein involved in cell-cell contacts) and MDH1 (cytosolic Malate dehydrogenase 1), revealed their role in early stages of autophagy during autophagosome formation. MPP7 was involved in the activation of YAP1 (a transcriptional coactivator in the Hippo pathway), which in turn promoted autophagy, whereas MDH1 was required for maintenance of the levels of the essential autophagy initiator serine-threonine kinase ULK1, and increased in the activity upon induction of autophagy. Our results provide a possible explanation for how autophagy is regulated by MPP7 and MDH1, which adds to our understanding of autophagy regulation in PDAC. SIGNIFICANCE: This study identifies and characterizes MPP7 and MDH1 as novel regulators of autophagy, which is thought to be responsible for pancreatic cancer cell survival.


Assuntos
Autofagia , Carcinoma Ductal Pancreático/patologia , Regulação Neoplásica da Expressão Gênica , Malato Desidrogenase/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proliferação de Células , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/genética , Proteínas de Membrana/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteínas de Sinalização YAP
7.
Transcription ; 9(2): 131-136, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28980873

RESUMO

Autophagy is an essential cellular process that degrades cytoplasmic organelles and components. Precise control of autophagic activity is achieved by context-dependent signaling pathways. Recent studies have highlighted the involvement of transcriptional programs during autophagic responses to various signals. Here, we summarize the current understanding of the transcriptional regulation of autophagy.


Assuntos
Autofagia , Transcrição Gênica , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Ciclo Celular , Código das Histonas , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional
8.
Front Oncol ; 7: 28, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28316954

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the few cancer types where the 5-year survival rate shows no improvement. Despite conflicting evidence, the majority of data points to an essential role for autophagy in PDAC growth and survival, in particular constitutively activated autophagy, can provide crucial fuel to PDAC tumor cells in their nutrient-deprived environment. Autophagy, which is required for cell homeostasis, can both suppress and promote tumorigenesis and tumor survival in a context-dependent manner. Protein by protein, the mystery of how PDAC abuses the cell's homeostasis system for its malignant growth has recently begun to be unraveled. In this review, we focus on how autophagy is responsible for growth and development of PDAC tumors and where autophagy and the mechanisms controlling it fit into PDAC metabolism. Understanding the range of pathways controlling autophagy and their interplay in PDAC could open the way for new therapeutic avenues.

9.
Structure ; 24(3): 437-47, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26876098

RESUMO

Members of the Toll-like receptor and interleukin-1 (IL-1) receptor families all signal via Toll/IL-1R (TIR) domain-driven assemblies with adaptors such as MyD88. We here combine the mammalian two-hybrid system MAPPIT and saturation mutagenesis to complement and extend crystallographic and nuclear magnetic resonance data, and reveal how TIR domains interact. We fully delineate the interaction sites on the MyD88 TIR domain for homo-oligomerization and for interaction with Mal and TLR4. Interactions between three sites drive MyD88 homo-oligomerization. The BB-loop interacts with the αE-helix, explaining how BB-loop mimetics inhibit MyD88 signaling. The αC'-helix interacts symmetrically. The MyD88 TIR domains thus assemble into a left-handed helix, compatible with the Myddosome death domain crystal structure. This assembly explains activation of MyD88 by Mal and by an oncogenic mutation, and regulation by phosphorylation. These findings provide a paradigm for the interaction of mammalian TIR domains.


Assuntos
Mutação , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/genética , Sítios de Ligação , Células HEK293 , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fator 88 de Diferenciação Mieloide/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Receptor 4 Toll-Like/metabolismo
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