Identification of novel cytochrome c releasing factor and analysis of its molecular function on apoptosis

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Mitochondria play an essential role in apoptosis by releasing apoptogenic factors including cytochrome c into the cytoplasm. To explore its molecular mechanisms, I planned two studies ; (1) Analysis of Bax translocation mechanisms, and (2) Identification of novel cytochrome c releasing factor.(1)Analysis of Bax translocation mechanisms- Bax, one of crucial proapoptotic Bcl2 family protein, is normally localized in the cytoplasm, and translocate to the mitochondria during apoptosis. To investigate the mechanisms of this translocation, we found that cytoplasmic protein 14-3-36 is normally bound to Bax in the cytoplasm, and that Bax dissociates from 14-3-36 during apoptosis to induce apoptotic changes in the mitochondria. In isolated mitochondria, immunodepletion of 14-3-36 from cell lysates enhanced integration of recombinant Bax into the mitochondrial membrane, and 14-3-36 inhibited Bax integration. These findings indicate that 14-3-39 plays a crucial role in negatively regulating Bax activity in living cells.(2) Identification of novel cytochrome c releasing factor- To identify the novel cytochrome c releasing factor, we purified this factor from irradiated thymus in mice. Addition gf irradiated, but not normal, thymus cytosol to mitochondria significantly induced cytochrome c release. Using FPLC column following amino acid. sequencing, we purified and identified this factor from apoptotic thymus cytosol. This recombinant protein actually induced cytochrome c release when added into mitochondria. These findings might imply that this factor is a novel cytochrome c releasing factor exerting on radiation-induced apoptosis.