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1.
J Biol Chem ; 292(47): 19209-19225, 2017 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-28972160

RESUMEN

The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control. A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag. We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates. We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14. The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases. Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant. We also report that a specific residue in tau, lysine 174, is critical for the IU1-47-mediated tau degradation by the proteasome. Finally, we show that IU1-47 stimulates autophagic flux in primary neurons. In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.


Asunto(s)
Embrión de Mamíferos/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Neuronas/metabolismo , Pirroles/farmacología , Ubiquitina Tiolesterasa/fisiología , Proteínas tau/metabolismo , Animales , Células Cultivadas , Citoplasma/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Pirroles/síntesis química , Ratas Sprague-Dawley , Ubiquitina/metabolismo , Ubiquitinación
2.
Immunity ; 44(6): 1299-311, 2016 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-27234056

RESUMEN

Mitochondrial respiration is regulated in CD8(+) T cells during the transition from naive to effector and memory cells, but mechanisms controlling this process have not been defined. Here we show that MCJ (methylation-controlled J protein) acted as an endogenous break for mitochondrial respiration in CD8(+) T cells by interfering with the formation of electron transport chain respiratory supercomplexes. Metabolic profiling revealed enhanced mitochondrial metabolism in MCJ-deficient CD8(+) T cells. Increased oxidative phosphorylation and subcellular ATP accumulation caused by MCJ deficiency selectively increased the secretion, but not expression, of interferon-γ. MCJ also adapted effector CD8(+) T cell metabolism during the contraction phase. Consequently, memory CD8(+) T cells lacking MCJ provided superior protection against influenza virus infection. Thus, MCJ offers a mechanism for fine-tuning CD8(+) T cell mitochondrial metabolism as an alternative to modulating mitochondrial mass, an energetically expensive process. MCJ could be a therapeutic target to enhance CD8(+) T cell responses.


Asunto(s)
Linfocitos T CD8-positivos/fisiología , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/inmunología , Adenosina Trifosfato/metabolismo , Animales , Respiración de la Célula , Células Cultivadas , Memoria Inmunológica , Interferón gamma/metabolismo , Activación de Linfocitos , Metaboloma , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/genética , Chaperonas Moleculares/genética , Fosforilación Oxidativa
3.
Mol Cell Biol ; 33(11): 2302-14, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23530063

RESUMEN

Mitochondria are the main engine that generates ATP through oxidative phosphorylation within the respiratory chain. Mitochondrial respiration is regulated according to the metabolic needs of cells and can be modulated in response to metabolic changes. Little is known about the mechanisms that regulate this process. Here, we identify MCJ/DnaJC15 as a distinct cochaperone that localizes at the mitochondrial inner membrane, where it interacts preferentially with complex I of the electron transfer chain. We show that MCJ impairs the formation of supercomplexes and functions as a negative regulator of the respiratory chain. The loss of MCJ leads to increased complex I activity, mitochondrial membrane potential, and ATP production. Although MCJ is dispensable for mitochondrial function under normal physiological conditions, MCJ deficiency affects the pathophysiology resulting from metabolic alterations. Thus, enhanced mitochondrial respiration in the absence of MCJ prevents the pathological accumulation of lipids in the liver in response to both fasting and a high-cholesterol diet. Impaired expression or loss of MCJ expression may therefore result in a "rapid" metabolism that mitigates the consequences of metabolic disorders.


Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Proteínas del Choque Térmico HSP40/genética , Metabolismo de los Lípidos/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Respiración de la Célula/genética , Colesterol/efectos adversos , Dieta , Complejo I de Transporte de Electrón/genética , Hígado Graso/genética , Femenino , Regulación de la Expresión Génica , Humanos , Membranas Intracelulares/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Rotenona/farmacología
4.
Mol Cell Biol ; 27(8): 2952-66, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17283040

RESUMEN

Methylation-controlled J protein (MCJ) is a newly identified member of the DnaJ family of cochaperones. Hypermethylation-mediated transcriptional silencing of the MCJ gene has been associated with increased chemotherapeutic resistance in ovarian cancer. However, the biology and function of MCJ remain unknown. Here we show that MCJ is a type II transmembrane cochaperone localized in the Golgi network and present only in vertebrates. MCJ is expressed in drug-sensitive breast cancer cells but not in multidrug-resistant cells. The inhibition of MCJ expression increases resistance to specific drugs by inducing expression of the ABCB1 drug transporter that prevents intracellular drug accumulation. The induction of ABCB1 gene expression is mediated by increased levels of c-Jun due to an impaired degradation of this transcription factor in the absence of MCJ. Thus, MCJ is required in these cells to prevent c-Jun-mediated expression of ABCB1 and maintain drug response.


Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Transportadores de Anión Orgánico/genética , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-jun/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Secuencia Conservada , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/ultraestructura , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/deficiencia , Proteínas del Choque Térmico HSP40/genética , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Transportadores de Anión Orgánico/metabolismo , Filogenia , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Vertebrados
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