RESUMEN
Misfolded proteins compromise cellular function and cause disease. How these proteins are detected and degraded is not well understood. Here we show that PML/TRIM19 and the SUMO-dependent ubiquitin ligase RNF4 act together to promote the degradation of misfolded proteins in the mammalian cell nucleus. PML selectively interacts with misfolded proteins through distinct substrate recognition sites and conjugates these proteins with the small ubiquitin-like modifiers (SUMOs) through its SUMO ligase activity. SUMOylated misfolded proteins are then recognized and ubiquitinated by RNF4 and are subsequently targeted for proteasomal degradation. We further show that PML deficiency exacerbates polyglutamine (polyQ) disease in a mouse model of spinocerebellar ataxia 1 (SCA1). These findings reveal a mammalian system that removes misfolded proteins through sequential SUMOylation and ubiquitination and define its role in protection against protein-misfolding diseases.
Asunto(s)
Degeneración Nerviosa/patología , Pliegue de Proteína , Proteolisis , Animales , Ataxina-1 , Ataxinas , Humanos , Ratones , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Proteína de la Leucemia Promielocítica , Complejo de la Endopetidasa Proteasomal , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismo , Sumoilación , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiología , Ubiquitina , UbiquitinaciónRESUMEN
An adequate cellular capacity to degrade misfolded proteins is critical for cell survival and organismal health. A diminished capacity is associated with aging and neurodegenerative diseases; however, the consequences of an enhanced capacity remain undefined. Here, we report that the ability to clear misfolded proteins is increased during oncogenic transformation and is reduced upon tumor cell differentiation. The augmented capacity mitigates oxidative stress associated with oncogenic growth and is required for both the initiation and maintenance of malignant phenotypes. We show that tripartite motif-containing (TRIM) proteins select misfolded proteins for proteasomal degradation. The higher degradation power in tumor cells is attributed to the upregulation of the proteasome and especially TRIM proteins, both mediated by the antioxidant transcription factor Nrf2. These findings establish a critical role of TRIMs in protein quality control, connect the clearance of misfolded proteins to antioxidant defense, and suggest an intrinsic characteristic of tumor cells.
Asunto(s)
Carcinogénesis/metabolismo , Carcinogénesis/patología , Pliegue de Proteína , Proteolisis , Animales , Antioxidantes/metabolismo , Autofagia , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Factor 2 Relacionado con NF-E2/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Oncogenes , Estrés Oxidativo , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia ArribaRESUMEN
p53 plays a central role in tumor suppression. It does so by inducing anti-proliferative processes as a response to various tumor-promoting stresses. p53 is regulated by the ubiquitin ligase Mdm2. The optimal function of Mdm2 requires Daxx, which stabilizes Mdm2 through the deubiquitinase Hausp/USP7 and also directly promotes Mdm2's ubiquitin ligase activity towards p53. The Daxx-Mdm2 interaction is disrupted upon DNA damage. However, both the mechanisms and the consequence of the Daxx-Mdm2 dissociation are not understood. Here we show that upon DNA damage Daxx is phosphorylated in a manner that is dependent on ATM, a member of the PI 3-kinase family that orchestrates the DNA damage response. The main phosphorylation site of Daxx is identified to be Ser564, which is a direct target of ATM. Phosphorylation of endogenous Daxx at Ser564 occurs rapidly during the DNA damage response and precedes p53 activation. Blockage of this phosphorylation event prevents the separation of Daxx from Mdm2, stabilizes Mdm2, and inhibits DNA damage-induced p53 activation. These results suggest that phosphorylation of Daxx by ATM upon DNA damage disrupts the Daxx-Mdm2 interaction and facilitates p53 activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/genética , Proteínas de Unión al ADN/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas Co-Represoras , Proteínas de Unión al ADN/genética , Humanos , Inmunoprecipitación , Chaperonas Moleculares , Datos de Secuencia Molecular , Neoplasias/genética , Neoplasias/patología , Proteínas Nucleares/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Serina/genética , Serina/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
TAp73 is a structural homologue of the pre-eminent tumour suppressor p53. However, unlike p53, TAp73 is rarely mutated, and instead is frequently overexpressed in human tumours. It remains unclear whether TAp73 affords an advantage to tumour cells and if so, what the underlying mechanism is. Here we show that TAp73 supports the proliferation of human and mouse tumour cells. TAp73 activates the expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). By stimulating G6PD, TAp73 increases PPP flux and directs glucose to the production of NADPH and ribose, for the synthesis of macromolecules and detoxification of reactive oxygen species (ROS). The growth defect of TAp73-deficient cells can be rescued by either enforced G6PD expression or the presence of nucleosides plus an ROS scavenger. These findings establish a critical role for TAp73 in regulating metabolism, and connect TAp73 and the PPP to oncogenic cell growth.