Assuntos
Adenoviridae/fisiologia , Espaço Intranuclear/fisiologia , Espaço Intranuclear/virologia , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Vírus de RNA/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Adenoviridae/genética , Animais , HIV-1/genética , HIV-1/fisiologia , Humanos , Camundongos , Complexos Multiproteicos/fisiologia , Proteína da Leucemia Promielocítica , Vírus de RNA/genética , Transdução de Sinais , Transcrição Gênica , Proteínas Virais/fisiologia , Replicação ViralRESUMO
Autoimmune regulator (AIRE) is responsible for the development of organ-specific autoimmune disease in a monogenic fashion. Rare and low levels of tissue expression together with the lack of AIRE-expressing cell lines have hampered a detailed analysis of the molecular dynamics of AIRE. Here we have established cell lines stably transfected with AIRE and studied the regulatory mechanisms for its subcellular expression. We found that nuclear body (NB) formation by AIRE was dependent on the cell cycle. Biochemical fractionation revealed that a significant proportion of AIRE is associated with the nuclear matrix, which directs the functional domains of chromatin to provide sites for gene regulation. Upon proteasome inhibition, AIRE NBs were increased with concomitant reduced expression in the cytoplasm, suggesting that subcellular targeting of AIRE is regulated by a ubiquitin-proteasome pathway. We also found that AIRE NBs compete for cAMP-response element-binding protein-binding protein/p300, a common coactivator of transcription, with the promyelocytic leukemia gene product. These results suggest that the transcriptional regulating activities of AIRE within a cell are controlled and organized in a spatiotemporal manner.
Assuntos
Regulação da Expressão Gênica , Frações Subcelulares/metabolismo , Fatores de Transcrição/genética , Animais , Ligação Competitiva , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Cisteína Endopeptidases/metabolismo , DNA Complementar/genética , Proteína p300 Associada a E1A , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Imuno-Histoquímica , Proteínas Luminescentes/genética , Camundongos , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Células NIH 3T3 , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase , Proteína da Leucemia Promielocítica , Complexo de Endopeptidases do Proteassoma , Proteínas Recombinantes de Fusão , Frações Subcelulares/ultraestrutura , Fatores de Tempo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Proteínas Supressoras de Tumor , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína AIRERESUMO
Autoimmune regulator (AIRE) gene mutation is responsible for the development of autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy, an organ-specific autoimmune disease with monogenic autosomal recessive inheritance. AIRE is predominantly expressed in medullary epithelial cells of the thymus and is considered to play important roles in the establishment of self-tolerance. AIRE contains two plant homeodomain (PHD) domains, and the novel role of PHD as an E3 ubiquitin (Ub) ligase has just emerged. Here we show that the first PHD (PHD1) of AIRE mediates E3 ligase activity. The significance of this finding was underscored by the fact that disease-causing missense mutations in the PHD1 (C311Y and P326Q) abolished its E3 ligase activity. These results add a novel enzymatic function for AIRE and suggest an indispensable role of the Ub proteasome pathway in the establishment of self-tolerance, in which AIRE is involved.