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

线粒体通过将包括细胞色素c在内的促凋亡因子释放到细胞质中，在细胞凋亡中发挥重要作用。为了探索其分子机制，我计划进行两项研究：(1)Bax易位机制的分析，(2)新的细胞色素c释放因子的鉴定。(1)Bax易位机制的分析-Bax是重要的促凋亡基因bcl2家族蛋白之一，通常定位于细胞质中，并在凋亡过程中移位到线粒体。为了研究这种易位的机制，我们发现细胞质蛋白14-3-36通常与细胞质中的Bax结合，并且在细胞凋亡过程中Bax与14-3-36解离，从而诱导线粒体发生凋亡变化。在分离的线粒体中，免疫耗竭14-3-36促进重组Bax整合到线粒体膜，14-3-36抑制Bax整合。这些发现表明14-3-39在负性调节活细胞中的Bax活性方面起着至关重要的作用。(2)新的细胞色素c释放因子的鉴定-为了鉴定新的细胞色素c释放因子，我们从辐射后的小鼠胸腺中纯化了该因子。外加gf照射，但不正常，胸腺胞浆对线粒体有显著的诱导细胞色素c释放。使用FPLC色谱柱跟踪氨基酸。通过测序，我们从凋亡的胸腺胞浆中纯化并鉴定了该因子。当这种重组蛋白加入线粒体中时，实际上会诱导细胞色素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发挥作用。

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.