Functional characterization of novel thioredoxin reductase and thioredoxin peroxidase in Drosophila

Missirlis, Fanis
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University of Guelph

Molecular oxygen is key to aerobic life, but is also converted into cytotoxic byproducts referred to as reactive oxygen species (ROS). In mammals, intracellular defense against ROS includes superoxide dismutase (Sod), catalase (Cat) and thiol-dependent redox systems, in which glutathione reductase (GR), thioredoxin reductase (TrxR) and the corresponding peroxidases are key enzymes. Mammalian TrxRs and glutathione peroxidases (GPxs) are selenium-containing enzymes. The fruit fly 'Drosophila' possesses most ROS-detoxifying enzymes reported for mammals with the possible exception of GR and GPx activities. This thesis presents an investigation of key 'Drosophila' antioxidant genes encoding TrxR-1 and thioredoxin peroxidase (TPx). I show that a single ' Drosophila' gene, termed 'Trxr-1', specifies cytoplasmic and mitochondrial non-selenocysteine-containing TrxRs that arise by alternative splicing. I generated transcript-specific mutants and used 'in vivo ' approaches to explore the biological activities of the two splicing variants by introducing the respective individual transgenes into ' Trxr-1' mutant flies. The results show that although the two respective TrxRs have similar biochemical properties, they cannot substitute for each other, 'in vivo. Trxr-1' null mutations result in larval death, whereas mutations causing reduced TrxR-1 activity reduce pupal eclosion and cause a severe shortening of the adult lifespan. I also provide genetic evidence for a functional interaction between TrxR-1, Sod1 and Cat, suggesting that the overall burden of ROS metabolism in 'Drosophila' is shared by the two defense systems. Finally, I report the 'in silico' identification of two non-selenium containing GPx-like genes in the ' Drosophila' genome and present an initial biochemical characterization of one of the two gene products. The results show that one of the GPx-like genes encodes a TPx rather than a GPx. Transgene-dependent overexpression of the TPx gene increases the resistance of individuals to experimentally-induced oxidative stress, but does not compensate for the loss of Cat, an enzyme which, like TPx, functions to eliminate intracellular hydrogen peroxide. Furthermore, transgene-derived overexpression of TPx in mutant flies lacking Sod1, an antioxidant enzyme which protects cells from superoxide radical toxicity, is detrimental. This contrasts to transgene-derived overexpression of Cat which can partially rescue the 'sod1' mutant. These observations indicate that TPx1 and Cat function in metabolically distinct pathways.

Drosophila, thioredoxin reductase, thioredoxin peroxidase, reactive oxygen species, superoxide dismutase