RESUMEN
The synthesis of proteins in the endoplasmic reticulum (ER) that exceeds the protein folding capacity of this organelle is a frequent cause of cellular dysfunction and disease. An example of such a disease is alpha-1-antitrypsin (A1AT) deficiency, caused by destabilizing mutations in this glycoprotein. It is considered that the mutant proteins are recognized in the ER by lectins and are subsequently degraded through the proteasome, leading to a deficiency in this enzyme in the afflicted patients. We previously established a Drosophila model of this disease by overexpressing the null Hong Kong (NHK) allele of this gene and found that the Drosophila lectin, ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2), can accelerate the degradation of A1AT when overexpressed. NHK is a rare allele, and in this study, we investigated in depth the mechanisms through which Drosophila EDEMs affect the degradation of the Z variant, which is the predominant disease allele. Specifically, we report that the Z allele does not activate ER stress signaling as prominently as the NHK allele, but similarly requires both Drosophila EDEM1 and EDEM2 for the degradation of the protein. We demonstrate that EDEMs are required for their ubiquitination, and without EDEMs, glycosylated A1AT mutants accumulate in cells. These results support the role of the EDEM-mediated ubiquitination of the alpha-1-antitrypsin Z (ATZ) allele, and establish a Drosophila model for the study of this protein and disease.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , alfa 1-Antitripsina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Análisis por Conglomerados , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Estrés del Retículo Endoplásmico , Técnicas de Silenciamiento del Gen , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Pliegue de Proteína , Proteolisis , Alineación de Secuencia , Ubiquitinación , alfa 1-Antitripsina/genéticaRESUMEN
Protein kinase A (PKA) phosphorylates diverse protein substrates to modulate their function. In this study, we found that PKA specifically phosphorylates the RD1 (repression domain 1) domain of nuclear receptor corepressor (NCoR). We demonstrated that the Serine-70 of NCoR is identified the critical amino acid for PKA-dependent NCoR phosphorylation. Importantly, we found that PKA-dependent phosphorylation enhances the nuclear translocation of NCoR. More importantly, the activation of PKA enhanced the repressive activity of NCoR in a reporter assay and potentiated the antagonist activity in the androgen receptor (AR)-mediated transcription. Taken together, these results uncover a regulatory mechanism by which PKA positively modulates NCoR function in transcriptional regulation in prostate cancer.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Co-Represor 1 de Receptor Nuclear/metabolismo , Neoplasias de la Próstata/enzimología , Transporte Activo de Núcleo Celular , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Mutación , Co-Represor 1 de Receptor Nuclear/genética , Fosforilación , Regiones Promotoras Genéticas , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/genética , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Serina , Transducción de Señal , Transcripción Genética , TransfecciónRESUMEN
Synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been reported to exhibit anti-invasive and anti-metastatic activities by suppressing the enzymatic activity of matrix metalloproteinase (MMP)-9, but the underlying mechanism remains unclear. Here, we show that 4-HPR blocks the activity of MMP-9 in two ways: by reducing phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 secretion and by suppressing cell invasion through the downregulation of MMP-9 gene transcription in MCF-7 breast cancer cells. 4-HPR inhibits the transcriptional activity of MMP-9 by reducing the DNA-binding activity of NF-κB on the MMP-9 promoter as well as by inhibiting the degradation of IκBα, leading to cytoplasmic accumulation of NF-κB. We also found that 4-HPR inhibits invasion and MMP-9 expression in the highly metastatic breast cancer cell line MDA-MB-231. Thus, 4-HPR might be a potent anti-invasive agent that works by suppressing MMP-9 expression via the NF-κB signaling pathway.