RESUMEN
Two homologs of the tumor suppressor p53, named p63 and p73, are each expressed from at least two start sites of mRNA synthesis, yielding full-length, transactivating (TA) isoforms, and also aminoterminally truncated (DeltaN) isoforms that act as antagonists to p53. The expression of TAp73-transcripts is induced by E2F and negatively regulated by transforming growth factor beta (TGFbeta). The DeltaNp73 promoter is induced by p53, resulting in negative feedback to control p53 activity. Here, we have analysed the expression of p63 in comparison with p73. In contrast to the induction of DeltaNp73, the expression of DeltaNp63 was reduced by p53 particularly in human keratinocytes, at the mRNA and protein levels. Accordingly, the 3' promoter of p73, but not that of p63, was activated by p53 in reporter assays. DeltaNp73 mRNA and DeltaNp73 protein, but not the p63 gene products, also accumulated when HaCat cells (lacking functional p53) were grown to high density. TAp73, but not TAp63, expression was suppressed by TGFbeta in these cells, and the TAp73, but not the TAp63, promoter was induced by E2F-1. Thus, in contrast to the functional similarities of their respective products, the expression levels of p63 and p73 are regulated by different mechanisms. This might be responsible for the discordant biological roles of p63 and p73 in development, as well as their deviant expression characteristics in cancer.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Proteínas de la Membrana , Proteínas Nucleares/genética , Fosfoproteínas/genética , Transactivadores/genética , Recuento de Células , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Genes Supresores de Tumor , Humanos , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de TumorRESUMEN
The 16 kDa cysteine-rich protein (16K) of tobacco rattle virus (TRV) is known to partially suppress RNA silencing in Drosophila cells. In this study, we show that 16K suppresses RNA silencing in green fluorescent protein (GFP)-transgenic Nicotiana benthamiana plants using an Agrobacterium-mediated transient assay. 16K slightly reduced the accumulation of short interfering RNAs (siRNA) of GFP, suggesting that the protein may interfere with the initiation and/or maintenance of RNA silencing. Deletion of either the N- or C-terminal part of 16K indicated that the entire 16K open reading frame (ORF) is necessary for its silencing suppression function. Pentapeptide insertion scanning mutagenesis (PSM) revealed that only two short regions of 16K tolerated five extra amino acid insertions without considerable reduction in its silencing suppression function. The tolerant regions coincide with sequence variability between tobravirus cysteine-rich proteins, indicating a strong functional and/or structural conservation of TRV 16K. Confocal laser scanning microscopy of transiently expressed 16K fusions to red fluorescent protein (RFP) revealed a predominant cytoplasmic localization and, in addition, a nuclear localization. In contrast, fusions of RFP with the N-terminal region of 16K localized exclusively to the cytoplasm, whereas fusions between RFP and the C-terminal region of 16K displayed an exclusive nuclear localization. Further analysis of 16K-derived peptide fusions demonstrated that the 16K C-terminal region contained at least two functional bipartite nuclear localization signals which were independently capable of nuclear targeting.
Asunto(s)
Virus de Plantas/fisiología , Virus ARN/fisiología , ARN Interferente Pequeño/antagonistas & inhibidores , Proteínas Virales/análisis , Secuencia de Aminoácidos , Fusión Artificial Génica , Línea Celular , Núcleo Celular/química , Citoplasma/química , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Insercional , Señales de Localización Nuclear , Interferencia de ARN , Rhizobium/genética , Alineación de Secuencia , Eliminación de Secuencia , Nicotiana/virología , Proteína Fluorescente RojaRESUMEN
The RNA genome of Plum pox virus (PPV) encodes one large polyprotein that is subsequently cleaved into mature viral proteins. One of the products of proteolytic processing, the 6K1 protein, has not yet been identified in vivo for any member of the genus Potyvirus. In this study, 6K1-specific polyclonal antiserum was raised against PPV 6K1 expressed in Escherichia coli as a translational fusion with the N terminus of avian troponin C and an unusual metal-binding cluster of troponin T-1. For detection of 6K1 in vivo, a pPPV-H6K1-NAT infectious clone was constructed, enabling concentration of histidine-tagged 6K1 by affinity chromatography. Affinity-purified 6K1 was detected in locally infected Nicotiana benthamiana leaves at 4, 7 and 14 days post-inoculation (d.p.i.) and, in addition, in systemically infected leaves at 14 d.p.i., 6K1 was detected exclusively as a protein of 6 kDa and no polyprotein precursors were identified with the raised anti-6K1 antiserum.