Abstract:
The synthesis of glutathione (GSH), a key intracellular antioxidant, requires the enzyme glutamate-cysteine ligase (GCL) which catalyzes the rate-limiting step in its formation. GCL is comprised of a catalytic subunit (GCLc) and modifier subunit (GCLm), with GCLc possessing all of the catalytic activity. GCLm, however, modulates the activity of the holoenzyme by increasing its affinity for glutamate and by decreasing the feedback inhibition by GSH. The studies presented herein provide evidence that upregulation of GCLm expression is part of the cell's defense against oxidative stress and describe the cloning and characterization of the mouse Gclm promoter in an attempt to elucidate mechanisms by which this occurs. Inducible expression of Gclm is found to be mediated by an antioxidant response element (ARE) located in its proximal promoter region while basal expression is mediated by an upstream AP-1 site. Additional evidence for involvement of the transcription factors Nrf1 and Nrf2 in Gclm expression is provided. Furthermore, the finding that oxidized LDL induces Gclm expression via the AR implies that the ARE is responsive to endogenous compounds and suggests a role for Gclm in the development of diseases for which oxidative stress is a contributing factor.