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

新型细胞色素C释放因子的鉴定及其对细胞凋亡的分子功能分析

基本信息

批准号：
12680695
负责人：
SHIMIZU Shigeomi
金额：
$ 2.56万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680695/
关键词：
apoptosis mitochondria Bax cytochrome c

项目摘要

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.

线粒体通过将包括细胞色素c在内的致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，我计划了两项研究：（1）Bax易位机制的分析，和（2）新的细胞色素c释放因子的鉴定。（1）Bax易位机制分析Bax是Bcl 2家族中重要的促凋亡蛋白，通常定位于细胞质，在细胞凋亡过程中，Bax易位到线粒体。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Bax在凋亡过程中从14-3-36解离以诱导线粒体中的凋亡变化。在分离的线粒体中，免疫耗竭14-3-36从细胞裂解物增强整合到线粒体膜的重组Bax，和14-3-36抑制Bax整合。这些发现表明14-3-39在负调节活细胞中Bax活性中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，我们从照射的小鼠胸腺中纯化了该因子。加辐射gf，但不正常，胸腺细胞质线粒体显着诱导细胞色素c的释放。使用FPLC柱跟踪氨基酸。测序，我们从凋亡胸腺胞质溶胶中纯化并鉴定了该因子。这种重组蛋白实际上诱导细胞色素c释放时，加入到线粒体。这些结果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。