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

线粒体通过向细胞质中释放细胞色素c等凋亡因子，在细胞凋亡过程中发挥重要作用。为了探索其分子机制，我计划了两项研究；(1)分析Bax易位机制；(2)鉴定新的细胞色素c释放因子。(1) Bax易位机制分析——Bax是促凋亡的关键Bcl2家族蛋白之一，通常定位于细胞质，在细胞凋亡过程中易位至线粒体。为了研究这种易位的机制，我们发现细胞质蛋白14-3-3 -36通常与细胞质中的Bax结合，而Bax在细胞凋亡过程中与14-3-3 -36分离，诱导线粒体发生凋亡变化。在分离的线粒体中，细胞裂解液中的14-3-36免疫缺失增强了重组Bax进入线粒体膜的整合，而14-3-36抑制了Bax的整合。这些结果表明14-3-39在负调控活细胞Bax活性中起着至关重要的作用。(2)新型细胞色素c释放因子的鉴定为了鉴定新型细胞色素c释放因子，我们从辐照小鼠胸腺中纯化了该因子。此外，gf辐照胸腺细胞液对线粒体的释放显著诱导细胞色素c的释放。采用高效液相色谱柱跟踪氨基酸。测序，我们从凋亡的胸腺细胞质中纯化并鉴定了该因子。当加入线粒体时，这种重组蛋白实际上诱导细胞色素c的释放。这些发现可能暗示该因子是一种新的细胞色素c释放因子，在辐射诱导的细胞凋亡中起作用。

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.