Twiddy Davina, Brown David G, Adrain Colin, Jukes Rebekah, Martin Seamus J, Cohen Gerald M, MacFarlane Marion, Cain Kelvin
Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, Leicester LE1 9HN, United Kingdom.
J Biol Chem. 2004 May 7;279(19):19665-82. doi: 10.1074/jbc.M311388200. Epub 2004 Mar 1.
The apoptosome is a large caspase-activating ( approximately 700-1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is approximately 135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9(1-130)) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9(1-130), demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6-8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.
凋亡小体是一种大型的半胱天冬酶激活复合物（约700 - 1400 kDa），当细胞色素c在线粒体依赖性凋亡性细胞死亡过程中释放时，它由凋亡蛋白酶激活因子-1（Apaf-1）和半胱天冬酶-9组装而成。核心支架蛋白Apaf-1约为135 kDa，包含半胱天冬酶募集结构域（CARD）、CED-4以及多个（13个）WD40重复结构域，这些结构域可能与多种未知的调节蛋白相互作用。为了鉴定此类蛋白，我们用dATP/细胞色素c激活THP.1裂解物，并使用蔗糖密度离心和基于亲和的方法纯化凋亡小体，以便通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF质谱）进行分析。首先，我们使用了一种含有半胱天冬酶-9（1 - 130）的CARD结构域的谷胱甘肽S-转移酶（GST）融合蛋白（GST-casp9(1-130)），当Apaf-1处于凋亡小体中时，它与Apaf-1的CARD结构域结合，并阻断半胱天冬酶-9的募集/激活。这种亲和纯化的凋亡小体复合物仅包含Apaf-1XL和GST-casp9(1-130)，表明Apaf-1的WD40和CED-4结构域不会稳定地结合其他胞质蛋白。接下来，我们使用抗半胱天冬酶-9单克隆抗体从细胞裂解物中免疫纯化天然活性凋亡小体复合物，该裂解物中细胞色素c、第二线粒体衍生的半胱天冬酶激活剂（Smac）或Omi/HtrA2的含量可忽略不计。这种凋亡小体复合物表现出低半胱天冬酶加工活性，并且包含四种稳定相关的蛋白，即Apaf-1、半胱天冬酶-9的前体p35/34形式、半胱天冬酶-3的前体p20形式、X连锁凋亡抑制蛋白（XIAP）以及细胞色素c，细胞色素c仅与该复合物短暂结合。然而，在含有Smac和Omi/HtrA2的裂解物中，纯化的凋亡小体复合物的半胱天冬酶加工活性增加了6 - 8倍，并且仅包含Apaf-1和半胱天冬酶-9的p35/p34加工亚基。在细胞凋亡过程中，Smac、Omi/HtrA2和细胞色素c同时从线粒体中释放，因此凋亡细胞中的功能性凋亡小体复合物可能主要由Apaf-1和加工后的半胱天冬酶-9组成。
