Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1
Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1
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DOI：
10.1074/jbc.c000405200
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发表时间：
2000-10-06
影响因子：
4.8
通讯作者：
Wang, XD
中科院分区：
文献类型：
--
作者：
Jiang, XJ;Wang, XD
我们报道了用高纯度的细胞色素c、重组的凋亡蛋白激活因子-1(APAF-1)和重组的proaspase-9对重组的proaspase-9进行的生化分析。通过核苷酸结合实验，我们发现APAF-1单独与dATP结合很弱，而与APAF-1的核苷酸结合被细胞色素c显著刺激，dATP与APAF-1的结合诱导形成多聚体APAF-1细胞色素c复合体。Procaspase-9还以细胞色素c依赖的方式协同促进dATP与APAF-1的结合，与凋亡体结合的dATP仍然是dATP，而不是dADP。一种非水解性的ATP类似物AD-PCP(β，γ-亚甲基腺苷5‘-三磷酸)能够代替dATP或ATP支持凋亡体的形成和caspase的激活。这些数据表明，APAF-1介导的caspase-9激活的关键事件是细胞色素c诱导的dATP与APAF-1的结合。
We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c, The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1 cytochrome c complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochrome c-dependent manner, The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, AD-PCP (beta,gamma-methylene adenosine 5'-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1.