The adapter protein apoptotic protease-activating factor-1 (Apaf-1) is proteolytically processed during apoptosis
The adapter protein apoptotic protease-activating factor-1 (Apaf-1) is proteolytically processed during apoptosis
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DOI：
10.1074/jbc.m101524200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Wesselborg, S
中科院分区：
文献类型：
--
作者：
Lauber, K;Appel, HAE;Wesselborg, S
Apoptotle蛋白酶激活因子-1（Apaf-1）是线粒体凋亡途径的关键调节因子，由3个功能区组成。(i)N-末端胱天蛋白酶募集结构域（CARD），其可以结合胱天蛋白酶原-9，（ii）CED-4样区，其能够自身寡聚化，和（iii）具有WD-40重复序列的调节C末端，其掩蔽CARD和CED-4区。在细胞凋亡过程中，细胞色素c和dATP可以减轻WD-40重复序列的抑制作用，从而使Apaf-1寡聚化和随后的招募和激活半胱氨酸蛋白酶原-9。在这里，我们报告说，不同的凋亡刺激诱导半胱天冬酶介导的切割Apaf-1成一个84 kDa的片段。通过将细胞裂解物与细胞色素c/dATP或半胱天冬酶-3孵育，但不与半胱天冬酶-6或半胱天冬酶-8孵育，在体外获得相同的Apaf-1片段。Apaf-1在N末端被切割，导致其CARD HI螺旋的去除。一个额外的切割位点位于WD-40重复序列内，即使在没有细胞色素c的情况下，也能使p84寡聚成类似于440-kDa Apaf-1多聚体。由于其CARD的部分丧失，p84多聚体缺乏半胱天冬酶-9或其它半胱天冬酶活性。因此，我们的数据表明，Apaf-1切割导致在凋亡过程中从凋亡小体释放半胱天冬酶。
Apoptotle protease-activating factor-1 (Apaf-1), a key regulator of the mitochondrial apoptosis pathway, consists of three functional regions. (i) an N-terminal caspase recruitment domain (CARD) that can bind to procaspase-9, (ii) a CED-4-like region enabling self-oligomerization, and (iii) a regulatory C terminus with WD-40 repeats masking the CARD and CED-4 region. During apoptosis, cytochrome c and dATP can relieve the inhibitory action of the WD-40 repeats and thus enable the oligomerization of Apaf-1 and the subsequent recruitment and activation of procaspase-9. Here, we report that different apoptotic stimuli induced the caspase-mediated cleavage of Apaf-1 into an 84-kDa fragment. The same Apaf-1 fragment was obtained in vitro by incubation of cell lysates with either cytochrome c/dATP or caspase-3 but not with caspase-6 or caspase-8. Apaf-1 was cleaved at the N terminus, leading to the removal of its CARD HI helix. An additional cleavage site was located within the WD-40 repeats and enabled the oligomerization of p84 into a similar to 440-kDa Apaf-1 multimer even in the absence of cytochrome c. Due to the partial loss of its CARD, the p84 multimer was devoid of caspase-9 or other caspase activity. Thus, our data indicate that Apaf-1 cleavage causes the release of caspases from the apoptosome in the course of apoptosis.