Since 1990, several genes have been identified that inhibit apoptosis, which is a common and widespread form of cell death. These genes include bcl-2 (a proto-oncogene that is overexpressed in many B-cell lymphomas), p35 (a baculovirus gene), ced-9 (a nematode developmental gene that is the homologue of bcl-2) and lmp-1 (an Epstein-Barr virus gene that enhances expressing of bcl-2). We discovered that bcl-2 inhibits neural cell death due to growth factors withdrawal, calcium ionophores, glucose withdrawal, membrane peroxidation, and free radical inducing agents (Zhong et al., 1993), suggesting that bc1-2 interacts with a biochemical step that is central to neural cell death. Moreover, bcl-2 inhibits both apoptotic and necrotic types of neural cell death via a reduction in cellular superoxide-driven Fenton chemistry. This represents the first understanding of the mechanism by which an anti-apoptotic gene functions, as well as the first demonstration that apoptosis is mediated by reactive oxygen species. Additionally, since Bcl-2 inhibits the formation of reactive oxygen species, the expression of bcl-2 may actually inhibit the aging process, as well as degenerative diseases. There are four likely mechanisms by which the protein Bcl-2 may inhibit the process of cell death mediated by Fenton-derived hydroxyl radicals: first, Bcl-2 may be a free radical scavenging protein. Second (and possibly in addition and to any scavenging activity), Bcl-2 may be a metal-binding protein. Third, Bcl-2 might be involved in the translocation of reduced glutathione into mitochondria. Fourth, Bcl-2 might inhibit the production of superoxide by interfering with the transfer of electrons from mitochondrial proteins (e.g., ubiquinone) to dioxygen, without actually forming a radical species itself. This proposal describes experiments that will distinguish between the four mechanistic possibilities described above, to extend our results to determine the biochemical mechanism by which bcl-2 inhibits cell death. Specifically, we propose to continue ongoing site-directed mutagenesis studies of bcl-2; to purify Bcl-2 protein from mutant yeast in which we have overexpressed bcl-2; to assay cytosolic and mitochondrial glutathione in bcl-2 expressing and control neural cells; to assay metal binding by Bcl-2; and, using electron spin resonance, to assess the ability of Bcl-2 to scavenge free radical species and to form a stable radical. The potential findings have important implications for the process of aging and degenerative disease.
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