There is a growing body of evidence indicating that oxidative stress in mammalian cells is one of the major causes of cancer, and considerable progress has been made in identifying and characterizing the chemistry of the possible oxidative processes and toxic oxygen metabolites that may be responsible. However, the precise identities of the targets and the reactive species responsible and the nature of the defense and repair systems in the cells are incompletely understood. The cytoplasmic antioxidants provide a primary line of defense against toxic oxidants originating either from the mitochondria or the cell exterior. This proposal describes experiments designed to elucidate the in vivo functions and antioxidant mechanisms of three major cytoplasmic antioxidant proteins, copper-zinc superoxide dismutase (CuZnSOD), catalase,,and copper metallothionein (CuMT) in the simple eucaryote Saccharomyces cerevisiae. The relationship between these water-soluble cytosolic antioxidant proteins and the lipid-soluble membrane antioxidant molecule ubiquinone will also be investigated. The yeast, S. cerevisiae, is an excellent model for studies of oxidative stress in eucaryotic cells. It is a simple eucaryote in which extensive genetics and molecular biology have been carried out, and cloned genes of most of the important antioxidant proteins and yeast strains with these genes lacking are available or easily constructed. The antioxidant proteins and their sub-cellular locations are very similar in yeast and humans; thus, yeast is an excellent system in which to probe their functions, regulation, and relationship to mutagenesis and disease, and, ultimately, to carcinogenic processes. Four questions will be addressed in these studies: (l) How does the cell sense the presence of oxygen in regulating the level of expression of these antioxidant proteins? (2) What is the role copper in the regulation and function of antioxidant proteins? At particular issue is whether copper acts as a prooxidant or an antioxidant in vivo. This question is based on our observation that Cu MT can play an important antioxidant role in cells lacking CuZnSOD in vivo. (3) How are the functions of these antioxidant proteins coupled in carrying out their functions? Here we will investigate the interrelation of these antioxidant proteins by studying mutant strains of yeast that either are lacking one or more of these proteins or that overproduce them. (4) Do the cytoplasmic antioxidant proteins function to protect the major membrane antioxidant, ubiquinol (coenzyme Q), from autoxidation? By successfully answering these questions, we will have made major progress in elucidating the mechanisms of cellular damage by active oxygen species and the role of these cytosolic antioxidant systems in protecting against such damage. We believe that our results will be highly relevant to some of the fundamental processes that lead to cancer.
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