RESUMEN
The Nrf1 (Nuclear factor E2-related factor 1) transcription factor performs a critical role in regulating cellular homeostasis. Using a proteomic approach, we identified Host Cell Factor-1 (HCF1), a co-regulator of transcription, and O-GlcNAc transferase (OGT), the enzyme that mediates protein O-GlcNAcylation, as cellular partners of Nrf1a, an isoform of Nrf1. Nrf1a directly interacts with HCF1 through the HCF1 binding motif (HBM), while interaction with OGT is mediated through HCF1. Overexpression of HCF1 and OGT leads to increased Nrf1a protein stability. Addition of O-GlcNAc decreases ubiquitination and degradation of Nrf1a. Transcriptional activation by Nrf1a is increased by OGT overexpression and treatment with PUGNAc. Together, these data suggest that OGT can act as a regulator of Nrf1a.
Asunto(s)
Factor C1 de la Célula Huésped/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Procesamiento Proteico-Postraduccional , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Expresión Génica , Glicosilación , Células HEK293 , Factor C1 de la Célula Huésped/química , Factor C1 de la Célula Huésped/genética , Humanos , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Factor Nuclear 1 de Respiración/química , Factor Nuclear 1 de Respiración/genética , Oximas/farmacología , Fenilcarbamatos/farmacología , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Activación Transcripcional/efectos de los fármacos , Transfección , UbiquitinaciónRESUMEN
The ubiquitin-proteasome system is important in maintaining protein homeostasis. NFE2-related factor 1 (Nrf1), a transcription factor in the cap 'n' collar basic-leucine zipper family, regulates expression of cytoprotective genes. It was previously shown that liver-specific knockout of Nrf1 (Nrf1LKO) leads to hepatic cell death, steatohepatitis and cancer. However, the mechanisms underlying these pathologies are not clear. Here, we report that Nrf1 is critical for proteasome gene expression in the liver. Liver-specific knockout of Nrf1 results in impaired basal and induced expression of proteasome genes, and diminished proteasome activity in hepatocytes. In addition, our findings demonstrated that endoplasmic reticulum stress signaling pathway was also activated in Nrf1LKO livers. Inhibition of proteasome activity leads to endoplasmic reticulum stress in Nrf1-deficient hepatocytes, prompting the development of steatosis in the liver. Our results indicate that Nrf1 plays an integral role in the maintenance of proteasome function in hepatocytes and in the prevention of liver steatosis development. Moreover, these results highlight an association between proteasome dysfunction, endoplasmic reticulum stress and steatosis.
Asunto(s)
Estrés del Retículo Endoplásmico , Hígado Graso/metabolismo , Hepatocitos/enzimología , Factor 1 Relacionado con NF-E2/fisiología , Complejo de la Endopetidasa Proteasomal/genética , Animales , Ácidos Borónicos , Bortezomib , Hígado Graso/inducido químicamente , Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hígado/metabolismo , Ratones , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/metabolismo , PirazinasRESUMEN
The ubiquitin-proteasome pathway plays an important role in the pathogenesis of neurodegeneration, but mechanisms controlling expression of components in this pathway remain poorly understood. Nuclear factor E2-related factor 1 (Nrf1) transcription factor has been shown to regulate expression of antioxidant and cytoprotective genes. To determine the function of Nrf1 in the brain, mice with a late-stage deletion of Nrf1 in neuronal cells were generated. Loss of Nrf1 leads to impaired proteasome function and neurodegeneration. Gene expression profiling and RT-PCR analysis revealed a coordinate down-regulation of various proteasomal genes including PsmB6, which encodes a catalytic subunit of the proteasome. Transcriptional analysis and chromatin immunoprecipitation experiments demonstrated that PsmB6 is an Nrf1 target gene. These findings reveal Nrf1 as a key transcriptional regulator required for the expression of proteasomal genes in neurons and suggest that perturbations of Nrf1 function may contribute to the pathogenesis of neurodegenerative diseases.
Asunto(s)
Encéfalo/patología , Regulación de la Expresión Génica , Factor 1 Relacionado con NF-E2/metabolismo , Degeneración Nerviosa/etiología , Complejo de la Endopetidasa Proteasomal/genética , Animales , Encéfalo/metabolismo , Ratones , Factor 1 Relacionado con NF-E2/deficiencia , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
In Saccharomyces cerevisiae, chemical or genetic inhibition of proteasome activity induces new proteasome synthesis promoted by the transcription factor RPN4. This ensures that proteasome activity is matched to demand. This transcriptional feedback loop is conserved in mammals, but its molecular basis is not understood. Here, we report that nuclear factor erythroid-derived 2-related factor 1 (Nrf1), a transcription factor of the cap "n" collar basic leucine zipper family, but not the related Nrf2, is necessary for induced proteasome gene transcription in mouse embryonic fibroblasts (MEFs). Promoter-reporter assays revealed the importance of antioxidant response elements in Nrf1-mediated upregulation of proteasome subunit genes. Nrf1(-/-) MEFs were impaired in the recovery of proteasome activity after transient treatment with the covalent proteasome inhibitor YU101, and knockdown of Nrf1 in human cancer cells enhanced cell killing by YU101. Taken together, our results suggest that Nrf1-mediated proteasome homeostasis could be an attractive target for therapeutic intervention in cancer.