RESUMEN
To elucidate the function of Omega class glutathione transferases (GSTs) (EC 2.5.1.18) in multicellular organisms, the GSTO-1 from Caenorhabditis elegans (GSTO-1; C29E4.7) was investigated. Disc diffusion assays using Escherichia coli overexpressing GSTO-1 provided a test of resistance to long-term exposure under oxidative stress. After affinity purification, the recombinant GSTO-1 had minimal catalytic activity toward classic GST substrates but displayed significant thiol oxidoreductase and dehydroascorbate reductase activity. Microinjection of the GSTO-1-promoter green fluorescent protein construct and immunolocalization by electron microscopy localized the protein exclusively in the intestine of all postembryonic stages of C. elegans. Deletion analysis identified an approximately 300-nucleotide sequence upstream of the ATG start site necessary for GSTO-1 expression. Site-specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression. Similarly, RNA interference (RNAi) of Elt-2 indicated the involvement of this gut-specific transcription factor in GSTO-1 expression. Transcriptional up-regulation under stress conditions of GSTO-1 was confirmed by analyzing promoter-reporter constructs in transgenic C. elegans strains. To investigate the function of GSTO-1 in vivo, transgenic animals overexpressing GSTO-1 were generated exhibiting an increased resistance to juglone-, paraquat-, and cumene hydroperoxide-induced oxidative stress. Specific silencing of the GSTO-1 by RNAi created worms with an increased sensitivity to several prooxidants, arsenite, and heat shock. We conclude that the stress-responsive GSTO-1 plays a key role in counteracting environmental stress.
Asunto(s)
Proteínas de Caenorhabditis elegans/clasificación , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Glutatión Transferasa/clasificación , Glutatión Transferasa/metabolismo , Estrés Oxidativo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Factores de Transcripción GATA/genética , Factores de Transcripción GATA/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Genes Reporteros/genética , Glutatión Transferasa/química , Glutatión Transferasa/genética , Humanos , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The nucleotide sequences for Brugia malayi and Wuchereria bancrofti GST have been submitted to EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under Accession Nos. Y12788 and AY195867.
