Glutathione S-Transferases: Novel Regulators of Cellular Stress and Apoptosis

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DESCRIPTION (provided by applicant): Glutathione S-transferases (GSTs) constitute a family of enzymes important in the detoxification of xenobiotics. They provide a defense against carcinogenesis, since they catalyze inactivation of known carcinogens; yet they contribute significantly to the development of resistance to cancer chemotherapy, since GST levels increase in tumors and the enzyme metabolizes key anticancer drugs. Interaction with other proteins is a new role for GSTs which yields regulation of diverse cellular functions. Thus, GSTs promote the cellular response to oxidative stress, since GSTpi activates the anti-oxidant enzyme 1-Cys peroxiredoxin (1-Cys Prx); and GSTs influence cell signaling through interactions with Jun N-terminal Kinase (JNK) and Apoptosis Signal-Regulating Kinase 1 (ASK1). With representatives of the pi, mu and alpha class GSTs, the most abundant mammalian GSTs, we will address the following questions: 1) What is the structural and chemical basis of activation by GSTpi of 1-Cys Prx? The heterodimeric complex between GST and 1-Cys Prx will be isolated, its kinetic properties for glutathione S-transferase and peroxiredoxin activities will be determined, and its physicochemical, as well as its substrate binding characteristics will be measured. 2) How does GSTpi inhibit the stress-activated kinase JNK, and what is the glutathione S-transferase activity of the complex? Is this effect specific for pi class GST or is inhibition of JNK a general property of GSTs? GST-JNK complexes will be isolated for rigorous characterization of its catalytic and biophysical properties. 3) What is the molecular basis of inhibition by mu class GST of the signal transduction protein Apoptosis Signal-Regulating Kinase 1? For isolated ASK1-GST complexes formed from ASK1 and GST (both wild-type and mutant), the molecular weight, conformation and re-dox state will be measured to elucidate the cause of inhibition of ASK1-mediated apoptosis and the effect of complex formation on GST activity. It is important to ascertain whether the glutathione and/or xenobiotic substrate sites are involved in the protein-protein functions of GST. These studies may lead to the rational design of pharmaceutical agents to prolong the lifetime of anticancer drugs by inhibiting particular GST sites without adversely affecting its other functional sites.