Age-related changes of glutathione homeostasis in Caenorhabditis elegans

Abstract

The de novo glutathione (GSH) synthesis pathway is highly conserved throughout all aerobic eukaryotic organisms. Intracellular de novo GSH synthesis involves two ATP-dependent enzyme-catalysed reactions; the rate-limiting and first reaction is mediated by glutamate cysteine ligase (GCL), which condenses glutamate and cysteine, to form gamma-glutamylcysteine (γ-GC). The second reaction involves the addition of glycine to γ-GC by glutathione synthetase (GS) to form GSH. During ageing, a progressive decline in cysteine availability or the expression and activity of the GCL enzyme has been implicated to contribute to a corresponding decline in cellular GSH levels. In mammals, the GCL enzyme is a heterodimeric holoenzyme composed of the catalytic (GCLC) and modifier (GCLM) subunits. Though GCLC contains all the substrate binding sites required for γ-GC production, its interaction with GCLM modulates its catalytic efficiency. The relative abundance of each subunit determines holoenzyme formation, with the GCLM subunit almost always being limiting. Tissues that are more vulnerable to oxidative stress, such as the brain, have been demonstrated in various rodent studies to have relatively lower levels of the GCLM subunit. The nematode, Caenorhabditis elegans, presents a useful alternative ageing model to investigate the changes in glutathione homeostasis due to its relatively short lifespan, and its expression of both the GCLC subunit (gcs-1 gene) and the recently identified GCLM orthologue (E01A2.1 gene). In this study, C. elegans were used to show that ageing leads to a pro-oxidising shift of the GSH/GSSG ratio with declines in GSH and γ-GC levels beginning from early adulthood. The regulatory role of the GCLM orthologue was also shown to maintain GSH levels in a manner similar to that of mammals, demonstrating C. elegans as an appropriate model to investigate the regulatory function of the GCLM subunit. Increasing the availability of the thiol precursor substrates, cysteine and γ-GC, were investigated to determine if the decline in GSH synthesis is a result of limited cysteine availability or the decline in the enzymatic production of γ-GC. Experiments comparing the efficacy of the cysteine prodrug, N-acetylcysteine (NAC), with exogenous γ-GC treatment were conducted to establish a bioavailability profile of both GSH precursors and to elucidate any phenotypic differences associated with improving longevity in GSH-compromised worms.