ABSTRACT
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.
Subject(s)
Caenorhabditis elegans Proteins/classification , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Glutathione Transferase/classification , Glutathione Transferase/metabolism , Oxidative Stress , Amino Acid Sequence , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/ultrastructure , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , GATA Transcription Factors/genetics , GATA Transcription Factors/metabolism , Gene Deletion , Gene Expression Regulation , Genes, Reporter/genetics , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Humans , Microscopy, Electron, Transmission , Microscopy, Immunoelectron , Molecular Sequence Data , Phylogeny , Promoter Regions, Genetic/genetics , RNA Interference , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sensitivity and Specificity , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
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.
