RESUMEN
Ovine adenovirus serotype 7 (OAdV), the prototype atadenovirus, has gene homologues for most mastadenovirus structural proteins but lacks proteins V and IX. Instead, OAdV has structural proteins of 32 and 42 kDa although the gene encoding the latter had not previously been identified. The presently reported studies of OAdV virions have now identified a minor structural polypeptide of approximately 40 kDa as the product of the L1 52/55-kDa gene and, more surprisingly, shown that the 42-kDa protein is encoded by LH3. This gene product was previously thought to be a homologue of mastadenovirus E1B 55 kDa, which is a multi-functional, non-structural protein that cooperates with E1A in cell transformation. The lack of transforming activity previously demonstrated for OAdV combined with a structural role for the LH3 product indicates that the protein has a different function in atadenoviruses. We discuss the abundance and likely core location of LH3 in the virion and the possible derivation of the E1B 55-kDa gene from the LH3 gene.
Asunto(s)
Proteínas E1B de Adenovirus/genética , Atadenovirus/genética , Mastadenovirus/genética , Proteínas Estructurales Virales/genética , Proteínas E1B de Adenovirus/química , Proteínas E1B de Adenovirus/fisiología , Secuencia de Aminoácidos , Animales , Atadenovirus/fisiología , Atadenovirus/ultraestructura , Genes Virales , Mastadenovirus/fisiología , Microscopía Electrónica , Datos de Secuencia Molecular , Peso Molecular , Homología de Secuencia de Aminoácido , Ovinos , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/fisiologíaAsunto(s)
Asparagina/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxígeno/metabolismo , Humanos , Hidroxilación , Unión Proteica , Proteínas Represoras/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Factores de Transcripción/metabolismoRESUMEN
Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF transcriptional activity is suppressed under normoxic conditions by hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators. Here we show that the protein FIH-1, previously shown to interact with HIF, is an asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O(2) to modify its substrate. Together with the recently discovered prolyl hydroxylases that regulate HIF stability, this class of oxygen-dependent enzymes comprises critical regulatory components of the hypoxic response pathway.
Asunto(s)
Asparagina/química , Oxigenasas de Función Mixta/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Transcripción Genética , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , Glutatión Transferasa/metabolismo , Humanos , Hidroxilación , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hierro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Oxígeno/metabolismo , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
The hypoxia-inducible factors (HIFs) 1alpha and 2alpha are key mammalian transcription factors that exhibit dramatic increases in both protein stability and intrinsic transcriptional potency during low-oxygen stress. This increased stability is due to the absence of proline hydroxylation, which in normoxia promotes binding of HIF to the von Hippel-Lindau (VHL tumor suppressor) ubiquitin ligase. We now show that hypoxic induction of the COOH-terminal transactivation domain (CAD) of HIF occurs through abrogation of hydroxylation of a conserved asparagine in the CAD. Inhibitors of Fe(II)- and 2-oxoglutarate-dependent dioxygenases prevented hydroxylation of the Asn, thus allowing the CAD to interact with the p300 transcription coactivator. Replacement of the conserved Asn by Ala resulted in constitutive p300 interaction and strong transcriptional activity. Full induction of HIF-1alpha and -2alpha, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively.