. This proposal focuses on the signal transduction mechanisms regulating the increase in de novo glutathione (GSH) synthesis that occurs during adaptation to oxidants and other electrophiles. Gamma-Glutamylcysteine synthetase (GCS), the rate limiting enzyme in de novo GSH synthesis, is composed of two subunits. Both GCS genes are transcriptionally up-regulated in response to stress from oxidants and electrophilic xenobiotics. Nonetheless, a mismatch between expression of the two GCS subunit proteins may result in altered kinetics and/or decreased stability of the enzyme. gamma-Glutamyl transpeptidase (GGT) is an exoenzyme that provides substrates for GSH synthesis by breaking down extracellular GSH. GGT enzymatic activity and mRNA transcription also increases in response to oxidants. While much has been learned over the past few years regarding mechanisms of GSH increases in adaptation, the signaling mechanisms that govern regulation of GCS and GGT on the transcriptional, post-transcriptional and post-translational levels remain largely unresolved. The investigators propose to test the following hypotheses in three aims: 1- that signaling for the transcriptional activation of GCS genes by electrophiles and H2O2-generating compounds occurs through overlapping pathways; 2- that the GCS regulatory subunit (GCS-LS) stabilizes the catalytic subunit against proteolysis as well as regulates the kinetics; 3- that GGT transcription by reactive oxygen species is mediated through activation specific signaling pathways and cis acting element(s) activated by H2O2. These hypotheses will be tested using the agents (2,3-dimethoxy-1,4-naphthoquinone (DMNQ), 4-hydroxynonenal (4HNE), and beta-naphthoflavone (BNF)) at concentrations and incubation times that cause increased transcription of both GCS subunits. Methods to be used include, dominant negative and constitutively active signaling components, kinase assays, in vivo footprinting, reporter construct, tetracycline regulated antisense transfection, and measurement of reactive oxygen species, glutathione oxidation and alpha-unsaturated aldehydes.
These aims will be carried out by using established cell models modified as needed to determine whether the conclusions are unique or generally applicable. The long-range goal of their research is an understanding of the regulation of GSH synthesis and the potential for its pharmacological manipulation to enhance synthesis of this essential cellular constituent.
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