The tripeptide glutathione (GSH; gamma- glutamylcysteinylglycine) is the most abundant non-protein thiol antioxidant within the cell and plays a central role in maintenance of cellular redox homeostasis and protection against oxidative injury. There is growing evidence that alterations in intracellular GSH levels play an important role in regulating apoptotic cell death. However, it is unknown whether GSH biosynthesis is dysregulated during apoptotic cell death. Glutamate cysteine ligase (GLCL) is a heterodimeric holoenzyme that catalyzes the rate-limiting step in GSH synthesis and consists of a catalytic subunit (GLCLC, 73 kDa) and a regulatory subunit (GLCLR, 31 kDa). Previous and preliminary studies indicate that GLCLC undergoes caspase-mediated cleavage during many forms of apoptosis. Interestingly, TGFbeta1-induced hepatocyte cell death results in both GLCLC cleavage and the loss of total GLCLC protein. Preliminary studies suggest that transcriptional and post-translational control mechanisms mediate these responses. These findings suggest that caspase-mediated cleavage of GLCLC and/or loss of GLCLC protein represents a novel mechanism regulating GLCL activity and GSH biosynthesis during TGFbeta1-induced apoptosis. We hypothesize that the cleavage and loss of GLCLC protein leads to the functional inactivation of GLCL during TGFbeta1-induced hepatocyte apoptosis. We believe that this loss of GSH biosynthetic capacity sensitizes cells to TGFbeta1-induced oxidative stress resulting in apoptotic cell death. The goal of Aim number 1 is to elucidate the functional effects of caspase-mediated GLCLC cleavage in vitro. For these studies multiple recombinant proteins will be utilized to examine the effects of GLCLC cleavage on GLCL holoenzyme formation and enzymatic activity.
Aims number 2 and number 3 will examine the transcriptional and post-translational mechanisms that mediate TGFbeta1-induced loss of GLCLC protein.
In Aim number 2 we will identify the murine GLCLC promoter region and transcription factor(s) responsible for TGFbeta1-induced suppression of GLCLC gene expression.
In Aim number 3 we will examine whether ubiquitination and proteasome-mediated degradation mediate TGFbeta1-induced loss of GLCLC protein.
In Aim number 4 we will examine whether overexpressing GLCL or a non-cleavable GLCLC enhances intracellular GSH biosynthesis and promotes resistance to TGFbeta1-induced cell death in cultured hepatocytes. The information gained from these studies will greatly enhance our understanding of the molecular mechanisms regulating GSH biosynthesis during TGFbeta1-induced hepatocyte apoptosis and will permit the development of a more comprehensive model regarding the dynamic regulation of GSH and redox homeostasis under various physiological and pathological conditions.
