Article 文章

Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade
细胞色素 c 和 dATP 依赖形成 Apaf-1/Caspase-9 复合物引发凋亡蛋白酶级联

Purification of Apaf-3 and Its Identification as Caspase-9
Apaf-3 的纯化及其鉴定为 Caspase-9

Caspase-3 activation was assayed by incubating in vitro–translated, ³⁵S-labeled, and affinity-purified caspase-3 with HeLa S-100 cytosolic fraction. Caspase-3 activation was initiated by the addition of dATP. The activated 20 kDa and 12 kDa fragments were separated from the 32 kDa precursor by SDS-PAGE (polyacrylamide gel electrophoresis) and visualized by phosphorimaging (25, 62).
Caspase-3 活化通过将体外翻译的、 ³⁵ S 标记的和亲和纯化的 caspase-3 与 HeLa S-100 细胞质分离物一起孵育来进行检测。Caspase-3 的活化是通过添加 dATP 来启动的。激活的 20 kDa 和 12 kDa 片段通过 SDS-PAGE（聚丙烯酰胺凝胶电泳）与 32 kDa 前体分离，并通过磷光成像（25, 62）可视化。

The separation of Apaf-1, -2, and −3 was initiated by applying HeLa cell S-100 to an SP-Sepharose column. Cytochrome c (Apaf-2) bound to the column while Apaf-1 and Apaf-3 flowed through it. Apaf-1 and Apaf-3 were further separated on a hydroxylapatite column, which bound Apaf-1 but not Apaf-3. Apaf-3 was further purified by the following six additional steps: Q-Sepharose ion-exchange chromatography, phenyl-sepharose hydrophobic-interaction chromatography, heparin-agarose ion-exchange chromatography, Superdex 200 gel filtration chromatography, Mono Q ion-exchange chromatography, and finally, glycerol gradient centrifugation. The results of the glycerol gradient are shown in Figure 1. After centrifugation, the fractions were collected from bottom to top and assayed for Apaf-3 activity by incubating each fraction with partially purified Apaf-1 (hydroxylapatite bound fraction), cytochrome c, and ³⁵S-labeled caspase-3 in the presence of dATP. Apaf-3 activity appeared in fractions 6–8 with a peak at fraction 7 (Figure 1A). The same fractions were subjected to SDS-PAGE followed by silver stain (Figure 1B). A single polypeptide band migrating just above the 45 kDa molecular weight marker coeluted with the Apaf-3 activity. The NH₂ terminus of this protein band was blocked to Edman degradation. To obtain protein sequence, the polypeptide band was blotted onto a PVDF membrane and subjected to tryptic digestion. The resulting tryptic peptides were separated by a reverse-phase HPLC column, and the sequences of two peptides were determined by Edman degradation. A database search revealed that the two peptide sequences, LSKPTLENLTPVVLR and TFDQLDISSLPTP, matched perfectly with amino acid residues 98–112 and 325–337, respectively, of human caspase-9/MCH6/ICE-LAP6 (Figure 1C, underlined).
Apaf-1、-2 和-3 的分离是通过将 HeLa 细胞 S-100 应用到 SP-Sepharose 柱上来启动的。细胞色素 c（Apaf-2）结合到柱上，而 Apaf-1 和 Apaf-3 则流过柱。Apaf-1 和 Apaf-3 在羟基磷灰石柱上进一步分离，羟基磷灰石结合 Apaf-1 但不结合 Apaf-3。Apaf-3 经过以下六个额外步骤进一步纯化：Q-Sepharose 离子交换色谱、苯基-Sepharose 疏水相互作用色谱、肝素琼脂糖离子交换色谱、Superdex 200 凝胶过滤色谱、Mono Q 离子交换色谱，最后是甘油梯度离心。甘油梯度的结果如图 1 所示。离心后，从底部到顶部收集各分数，并通过将每个分数与部分纯化的 Apaf-1（羟基磷灰石结合分数）、细胞色素 c 和标记有 S 的半胱氨酸蛋白酶-3 在存在 dATP 的情况下孵育来检测 Apaf-3 活性。Apaf-3 活性出现在第 6-8 分数，第 7 分数达到峰值（图 1A）。相同的分数经过 SDS-PAGE 后进行银染（图 1B）。一个单一的多肽带，迁移至 45 kDa 分子量标记物上方，与 Apaf-3 活性共同出现。这个蛋白带的 NH1 端被阻断以进行 Edman 降解。为了获得蛋白序列，多肽带被转印到 PVDF 膜上，并进行胰蛋白酶消化。通过反相 HPLC 柱分离产生的胰蛋白酶肽段，通过 Edman 降解确定了两个肽段的序列。数据库搜索显示，这两个肽段序列 LSKPTLENLTPVVLR 和 TFDQLDISSLPTP 与人类半胱氨酸蛋白酶-9/MCH6/ICE-LAP6 的氨基酸残基 98-112 和 325-337 完全匹配（图 1C，下划线）。

1. Download : Download high-res image (227KB)
   下载：下载高分辨率图像（227KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 1. Glycerol Gradient Purification of Apaf-3 and Its Identification as Caspase-9/Mch6/ICE-LAP6
图 1. 甘油梯度纯化 Apaf-3 及其鉴定为 Caspase-9/Mch6/ICE-LAP6

The Apaf-3 activity was purified through the glycerol gradient step as described in Experimental Procedures.
Apaf-3 活性经甘油梯度步骤纯化，如实验程序中所述。

(A) Aliquots of 1 μl of glycerol gradient fractions were incubated with aliquots of 4 μl of Apaf-1 fraction bound to hydroxylapatide column (Zou et al. 1997), 1 μl (0.2 μg) of cytochrome c, 3 μl of in vitro–translated, ³⁵S-labeled, and affinity-purified Caspase-3, and 1 mM dATP at 30°C for 1 hr in a final volume of 20 μl of buffer A. After 1 hr at 30°C, the samples were subjected to 15% SDS-PAGE and transferred to a nitrocellulose filter. The filter was exposed to a phosphorimaging plate for 16 hr at room temperature.
（A）取 1μl 甘油梯度分离物，与 4μl 结合到羟基磷灰石柱的 Apaf-1 分离物（Zou 等，1997 年），1μl（0.2μg）细胞色素 c，3μl 体外翻译的、 ³⁵ S-标记的和亲和纯化的 Caspase-3，以及 1mM dATP，在 30°C 下在 20μl 缓冲液 A 中孵育 1 小时。在 30°C 下孵育 1 小时后，样品经过 15% SDS-PAGE 并转移到硝酸纤维素滤膜。将滤膜暴露在室温下的荧光成像板上 16 小时。

(B) Aliquots (30 μl) of the indicated glycerol gradient fractions were subjected to 8% SDS-PAGE, and the gel was subsequently stained with silver using a Silver Stain Plus kit from Bio-Rad.
(B) 指示的甘油梯度分离物的等分（30 μl）经过 8% SDS-PAGE，然后用 Bio-Rad 的 Silver Stain Plus 套件进行银染色。

(C) The Apaf-3 peak fractions from the Mono Q column as described in the Experimental Procedures were subjected to 8% SDS-PAGE and electroblotted onto a PVDF membrane. The 46 kDa polypeptide band correlated with Apaf-3 activity was excised from the membrane and subjected to trypsin digestion. The resulting peptides were separated by a reverse-phase HPLC column, and the amino acids sequences of two individual peptides (underlined) were determined by Edman degradation in an Applied Biosystems sequencer. The amino acid sequence of caspase-9 was as reported in Srinivasula et al. 1996a. The conserved active site pentapeptide QACGG is boxed. The asterisk denotes the amino acid residue after which the cleavage occurs during caspase-9 activation (Srinivasula et al. 1996a).
(C) 从 Mono Q 柱中得到的 Apaf-3 峰值分离物按照实验程序描述进行了 8% SDS-PAGE，并电泳转移到 PVDF 膜上。与 Apaf-3 活性相关的 46 kDa 多肽带从膜上切下，并进行胰蛋白酶消化。产生的肽段通过反相 HPLC 柱分离，两个单独肽段（下划线）的氨基酸序列通过 Applied Biosystems 测序仪中的 Edman 降解确定。Caspase-9 的氨基酸序列如 Srinivasula 等人 1996a 年所述。保守的活性位点五肽 QACGG 被方框标记。星号表示在 Caspase-9 激活期间发生剪切的氨基酸残基（Srinivasula 等人 1996a 年）。

Reconsititution of Caspase-3 Activation with Purified Components
用纯化组分重组 Caspase-3 激活

Figure 2A shows the reconstitution of the caspase-3 activation reaction using three purified proteins: Apaf-1, cytochrome c, and Apaf-3. No single component nor any combination of two was sufficient to activate caspase-3 (lanes 1–6). All three proteins plus dATP were required to cleave caspase-3 (lane 7).
图 2A 显示了使用三种纯化蛋白质：Apaf-1、细胞色素 c 和 Apaf-3 重新构成 caspase-3 激活反应。没有单个组分或任何两种组合足以激活 caspase-3（第 1-6 条道）。需要所有三种蛋白质加上 dATP 才能裂解 caspase-3（第 7 条道）。

1. Download : Download high-res image (253KB)
   下载：下载高分辨率图像（253KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 2. Reconstitution of Caspase-3 Activation with Purified Components
图 2. 用纯化组分重构 Caspase-3 活化

Aliquots (2 μl) of in vitro–translated, ³⁵S-labeled, and affinity–purified caspase-3 were incubated at 30°C for 1 hr with aliquots of 2 μl Apaf-1 purified through the Mono Q column step as described in Zou et al. 1997 (lanes 1, 4, 5, and 7), or 1 μl (0.3 μg) cytochrome c (lanes 2, 4, 6, and 7), or 2 μl of purified Apaf-3 (12.5 ng) (lanes 3 and 5–7) in a final volume of 20 μl buffer A supplemented with 1 mM dATP and 1 mM MgCl₂. In lane 8, Apaf-1, cytochrome c and Apaf-3 were incubated as described above in the absence of in vitro-translated ³⁵S-labeled caspase-3. After incubation, samples were subjected to 15% SDS-PAGE and transferred to a nitrocellulose filter.
在 30°C 下，将体外翻译的 ³⁵ S 标记和纯化的 caspase-3（2 μl）与通过 Mono Q 柱步骤纯化的 Apaf-1（如 Zou 等人 1997 年描述的，1, 4, 5 和 7 道），或 1 μl（0.3 μg）细胞色素 c（2, 4, 6 和 7 道），或 2 μl 纯化的 Apaf-3（12.5 ng）（3 和 5-7 道）在含 1 mM dATP 和 1 mM MgCl ₂ 的 20 μl 缓冲液 A 中孵育 1 小时。在第 8 道，Apaf-1、细胞色素 c 和 Apaf-3 在没有体外翻译的 ³⁵ S 标记 caspase-3 的情况下进行了上述孵育。孵育后，样品经过 15% SDS-PAGE 分离并转移到硝酸纤维素滤膜。

(A) The filter was exposed to a phosphorimaging plate for 2 hr at room temperature.
（A）过滤器在室温下暴露在荧光成像板上 2 小时。

(B) The same filter was probed with a polyclonal anti-caspase-9 antibody generated as described in the Experimental Procedures. The antigen/antibody complex was visualized by an ECL method as described in the Experimental Procedure. The filter was exposed to a Kodak X-Omat AR X-ray film for 10 s.
(B) 使用在实验程序中描述的方法生成的多克隆抗 caspase-9 抗体探测相同的过滤器。 使用实验程序中描述的 ECL 方法可视化抗原/抗体复合物。 过滤器暴露在柯达 X-Omat AR X 射线胶片上，曝光时间为 10 秒。

Using a polyclonal antibody generated against a recombinant caspase-9 fusion protein, we examined the cleavage of caspase-9 during the caspase-3 activation reaction by immunoblot analysis (Figure 2B). Apaf-3 was recognized by the anti-caspase-9 antibody, confirming that Apaf-3 is caspase-9 (lanes 3 and 5–8). Caspase-9 was activated in the presence of Apaf-1, cytochrome c, and dATP regardless of whether caspase-3 was present (lanes 7 and 8), indicating that caspase-9 activation is independent of caspase-3 activity.
使用针对重组 caspase-9 融合蛋白生成的多克隆抗体，我们通过免疫印迹分析检测了 caspase-3 激活反应过程中 caspase-9 的裂解（图 2B）。Apaf-3 被抗 caspase-9 抗体识别，证实 Apaf-3 是 caspase-9（第 3 和第 5-8 条）。无论是否存在 caspase-3，Apaf-1、细胞色素 c 和 dATP 的存在都会激活 caspase-9（第 7 和第 8 条），表明 caspase-9 的激活与 caspase-3 的活性无关。

Caspase-9 Is Required for Caspase-3 Activation
Caspase-9 对 Caspase-3 的激活是必需的

To further demonstrate that caspase-9 is upstream of caspase-3, we immunodepleted caspase-9 from the HeLa cell S-100 fraction using the antibody against caspase-9. As shown in Figure 3, S100 depleted of either caspase-9 or cytochrome c failed to activate caspase-3 (lanes 3, 4, 7, and 8). Caspase-3 was not depleted by the antibodies against caspase-9 or cytochrome c since the caspase-3 precursor was present at approximately the same level in all reactions (Figure 3B). Addition of purified Apaf-3 or cytochrome c to extracts depleted of the respective proteins restored the cleavage of caspase-3 (lanes 5, 6, 9, and 10). The endogenous caspase-3 precursor present in these extracts was cleaved in the same fashion as the ³⁵S labeled caspase-3 (Figure 3B).
为了进一步证明 caspase-9 位于 caspase-3 的上游，我们使用针对 caspase-9 的抗体从 HeLa 细胞 S-100 分离物中免疫去除了 caspase-9。如图 3 所示，去除了 caspase-9 或细胞色素 c 的 S100 无法激活 caspase-3（第 3、4、7 和 8 条带）。由于 caspase-3 前体在所有反应中大致处于相同水平，因此 caspase-3 不会被针对 caspase-9 或细胞色素 c 的抗体去除（图 3B）。向去除了相应蛋白质的提取物中添加纯化的 Apaf-3 或细胞色素 c 可以恢复 caspase-3 的裂解（第 5、6、9 和 10 条带）。这些提取物中存在的内源性 caspase-3 前体以与标记为 S 的 caspase-3 相同的方式被裂解（图 3B）。

1. Download : Download high-res image (266KB)
   下载：下载高分辨率图像（266KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 3. Caspase-9 and Cytochrome c Are Required for Caspase-3 Activation
图 3. Caspase-9 和细胞色素 c 是 Caspase-3 激活所必需的

Depletion of caspase-9 from HeLa cell S-100 was performed as described in the Experimental Procedures. Depletion of cytochrome c was as described in Liu et al. 1996b.
从 HeLa 细胞 S-100 中去除 caspase-9 是按照实验程序描述的进行的。细胞色素 c 的去除是根据 Liu 等人 1996b 的描述进行的。

(A) Aliquots (50 μg) of HeLa cell S-100 (lanes 1 and 2), or Hela S-100 immunodepleted of caspase-9 (lanes 3–6), or Hela S-100 immunodepleted of cytochrome c (lanes 7–10) were incubated with aliquots (3 μl) of in vitro–translated, ³⁵S-labeled caspase-3 in the absence (lanes 1, 3, 5, 7, and 9) or presence (lanes 2, 4, 6, 8, and 10) of 1 mM dATP in a final volume of 20 μl of buffer A. Aliquots of 6 μl of Apaf-3 (37.5 ng) purified to the Mono Q step (lanes 5 and 6), or 0.2 μg purified cytochrome c (lanes 9 and 10) were supplemented to the indicated reactions. After incubating at 30°C for 1 hr, the samples were subjected to 15% SDS-PAGE, and the gel was subsequently transferred to a nitrocellulose filter. The filter was exposed to film for 3 days at −80°C.
(A) HeLa 细胞 S-100 的等分样品（50 μg）（lane 1 和 2），或去除 caspase-9 的 HeLa S-100（lane 3–6），或去除细胞色素 c 的 HeLa S-100（lane 7–10）与体外翻译的、 ³⁵ S-标记的 caspase-3 的等分样品（3 μl）在缺失（lane 1, 3, 5, 7, 和 9）或存在（lane 2, 4, 6, 8, 和 10）1 mM dATP 的情况下在缓冲液 A 的最终体积为 20 μl 的条件下孵育。6 μl 经纯化至 Mono Q 步骤的 Apaf-3（37.5 ng）（lane 5 和 6），或 0.2 μg 纯化的细胞色素 c（lane 9 和 10）被添加到指定的反应中。在 30°C 孵育 1 小时后，样品经过 15% SDS-PAGE，然后凝胶被转移到硝酸纤维滤纸上。滤纸在-80°C 下曝光 3 天。

(B) Same reactions were performed as in (A) except the in vitro–translated, ³⁵S-labeled caspase-3 was omitted. The samples were then subjected to 15% SDS-PAGE followed by electroblotting to a nitrocellulose filter. The filter was probed with 25 μg of a monoclonal antibody to caspase-3 (Transduction Laboratories). The antigen/antibody complexes were visualized by an ECL method as described in Experimental Procedures. The filter was exposed to Kodak X-OMAT X-ray film for 10 min.
(B) 与（A）中相同的反应进行，只是省略了体外翻译的， ³⁵ S-标记的 caspase-3。然后将样品经过 15% SDS-PAGE 电泳，随后电转印到硝酸纤维素滤膜上。滤膜用 25 μg 单克隆抗体对 caspase-3（Transduction Laboratories）进行探测。抗原/抗体复合物通过实验程序中描述的 ECL 方法可视化。滤膜曝露在柯达 X-OMAT X 射线胶片上 10 分钟。

Activated Caspase-9 Cleaves Caspase-3
活化的 Caspase-9 裂解 Caspase-3

The above data indicate that caspase-9 is the upstream caspase in a protease cascade that activates caspase-3. To demonstrate that active caspase-9 cleaves caspase-3, we generated recombinant active caspase-9 enzyme by overexpressing it in bacteria.
上述数据表明，caspase-9 是激活 caspase-3 的蛋白酶级联中的上游 caspase。为了证明活性 caspase-9 裂解 caspase-3，我们通过在细菌中过表达来生成重组活性 caspase-9 酶。

As shown in Figure 4, activated caspase-9 cleaves and activates caspase-3 in as early as 15 min. This implies that, once caspase-9 becomes activated, it will initiate a protease cascade leading to the rapid activation of caspsase-3, a major caspase activity in cells undergoing apoptosis (9, 27, 46). The enzymatic activity of caspase-9 can be inhibited by zVAD-fmk, a general caspase inhibitor that blocks cellular apoptotic response (data not shown).
如图 4 所示，激活的 caspase-9 在 15 分钟内就会裂解并激活 caspase-3。这意味着一旦 caspase-9 被激活，它将启动蛋白酶级联，导致 caspase-3 的快速激活，这是细胞凋亡中的主要 caspase 活性。caspase-9 的酶活性可以被 zVAD-fmk 抑制，这是一种阻断细胞凋亡反应的通用 caspase 抑制剂（数据未显示）。

1. Download : Download high-res image (84KB)
   下载：下载高分辨率图像（84KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 4. Caspase-9-Mediated Cleavage of Caspase-3
图 4. Caspase-9 介导的 Caspase-3 裂解

Active caspase-9 was prepared as described in the Experimental Procedures. Aliquots (10 ng) of active caspase-9 were incubated with aliquots (0.5 μl) of in vitro–translated, ³⁵S-labeled, and affinity-purified caspase-3 precursor for the indicated times at 37°C. The samples were subsequently subjected to 15% SDS-PAGE and exposed to X-ray film for 12 hr at room temperature.
活性 caspase-9 按照实验程序制备。活性 caspase-9 的小分子（10 ng）与体外翻译的、 ³⁵ S 标记的、亲和纯化的 caspase-3 前体的小分子（0.5 μl）在 37°C 下按照指定时间孵育。样品随后经过 15% SDS-PAGE 分离，并在室温下暴露于 X 射线胶片上 12 小时。

Evidence that Hydrolysis of dATP or ATP Is Required for Caspase-9 Activation
dATP 或 ATP 的水解是 Caspase-9 激活所必需的证据

Using only purified components, we reconstituted the caspase-3 activation reaction in the presence of varying concentrations of either dATP or ATP (Figure 5). Caspase-9 and caspase-3 were both activated in the presence of 1 μM dATP (lane 3, panels A and B). Similar activation occurred in the presence of ATP, but a much higher concentration of ATP (1 mM) was required (lane 7). This result is consistent with our previous observation that dATP is the preferred nucleotide for activation (Liu et al. 1996b). Neither caspase-9 nor caspase-3 was cleaved in the presence of the nonhydrolyzable ATP analogs, AMP-PNP or ATP-γ-S, suggesting that hydrolysis is required for the reaction (lanes 8–13). dADP, a nucleotide that substitutes for dATP in the crude system (Liu et al. 1996b), did not function in the purified system (data not shown). We suggest that dADP was converted to dATP in the crude system.
仅使用纯化的组分，我们在存在不同浓度的 dATP 或 ATP 的情况下重组了 caspase-3 激活反应（图 5）。在 1μM dATP 存在的情况下，caspase-9 和 caspase-3 均被激活（第 3 条，面板 A 和 B）。在 ATP 存在的情况下也发生了类似的激活，但需要更高浓度的 ATP（1mM）（第 7 条）。这个结果与我们先前观察到的 dATP 是首选核苷酸的观察一致（Liu 等，1996b）。在存在不可水解的 ATP 类似物 AMP-PNP 或 ATP-γ-S 的情况下，既不会裂解 caspase-9 也不会裂解 caspase-3，这表明水解是反应所必需的（第 8-13 条）。dADP 是一种在粗制系统中替代 dATP 的核苷酸（Liu 等，1996b），在纯化系统中无法发挥作用（未显示数据）。我们认为 dADP 在粗制系统中被转化为 dATP。

1. Download : Download high-res image (277KB)
   下载：下载高分辨率图像（277KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 5. Titration of dATP, ATP, and Nonhydrolyzable ATP Analogs in the Activation of Caspase-3 and Caspase-9
图 5. 在激活 Caspase-3 和 Caspase-9 过程中，对 dATP、ATP 和不可水解 ATP 类似物进行滴定

Aliquots (2 μl) of Apaf-1 purified through the Mono Q column step as described in Zou et al. 1997 were incubated at 30°C for 1 hr with aliquots of 2 μl of purified Apaf-3 (12.5 ng), 1 μl (0.3 μg) cytochrome c, and 2 μl of in vitro–translated, ³⁵S-labeled, and affinity-purified caspase-3 in a final volume of 20 μl of buffer A in the absence of nucleotides (lane 1), or in the presence of increasing concentrations of dATP (lanes 2, 3, and 4), ATP (lanes 5, 6, and 7), AMP-PNP (lanes 8, 9, and 10), or ATP-γ-S ( lanes 11, 12, and 13). Samples were subjected to 15% SDS-PAGE and transferred to a nitrocellulose filter.
Aliquots（2μl）经过 Mono Q 柱步骤纯化的 Apaf-1（如 Zou 等人 1997 年所述）在 30°C 下与纯化的 Apaf-3（12.5 ng，2μl）、1μl（0.3μg）细胞色素 c 和体外翻译的、 ³⁵ S-标记的、亲和纯化的 caspase-3 的 2μl 孵育 1 小时，最终体积为 20μl 的缓冲液 A 中，不含核苷酸（lane 1），或在存在不同浓度的 dATP（lane 2、3 和 4）、ATP（lane 5、6 和 7）、AMP-PNP（lane 8、9 和 10）或 ATP-γ-S（lane 11、12 和 13）的情况下。样品经过 15% SDS-PAGE 分离并转移到硝酸纤维素滤膜。

(A) The filter was exposed to a phosphorimaging plate for 2 hr at room temperature.
（A）过滤器在室温下暴露在荧光成像板上 2 小时。

(B) The same filter was probed with a polyclonal anti-caspase-9 antibody generated as described in the Experimental Procedures. The antigen/antibody complex was visualized by an ECL method as described in the Experimental Procedure. The filter was exposed to a Kodak X-Omat AR X-ray film for 10 s.
(B) 使用在实验程序中描述的方法生成的多克隆抗 caspase-9 抗体探测相同的过滤器。 使用实验程序中描述的 ECL 方法可视化抗原/抗体复合物。 过滤器暴露在柯达 X-Omat AR X 射线胶片上，曝光时间为 10 秒。

Apaf-1 and Caspase-9 Form a Complex in the Presence of dATP and Cytochrome c
Apaf-1 和 Caspase-9 在存在 dATP 和细胞色素 c 的情况下形成复合物

To further explore the mechanism of caspase-9 activation, we studied the interaction of caspase-9 with Apaf-1 by coimmunoprecipitation using the antibody against caspase-9. The precipitates were analyzed by immunoblotting with an antibody against Apaf-1 (Zou et al. 1997). As shown in Figure 6A, the anti-caspase-9 antibody precipitated Apaf-1 in the presence of cytochrome c, dATP and Apaf-3 (lane 10). Preimmune serum from the same animal did not precipitate Apaf-1 (lane 9). Omission of dATP (lanes 7–8), cytochrome c (lanes 3–6), Apaf-3 (lanes 11–12), or Apaf-1 (lanes 1–2) obliterated coimmunoprecipitation of Apaf-1.
为进一步探索 caspase-9 激活机制，我们通过共免疫沉淀使用针对 caspase-9 的抗体研究了 caspase-9 与 Apaf-1 的相互作用。通过使用针对 Apaf-1 的抗体进行免疫印迹分析沉淀物（Zou 等人，1997 年）。如图 6A 所示，抗 caspase-9 抗体在存在细胞色素 c、dATP 和 Apaf-3 的情况下沉淀了 Apaf-1（第 10 条道）。同一动物的未免疫血清未沉淀 Apaf-1（第 9 条道）。省略 dATP（第 7-8 条道）、细胞色素 c（第 3-6 条道）、Apaf-3（第 11-12 条道）或 Apaf-1（第 1-2 条道）会消除 Apaf-1 的共免疫沉淀。

1. Download : Download high-res image (260KB)
   下载：下载高分辨率图像（260KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 6. Formation of Apaf-1/Caspase-9 Complex
图 6. Apaf-1/Caspase-9 复合物的形成

(A) Apaf-1 and Apaf-3 were seperated by a hydroxyapatite column as described in Zou et al. 1997 and used for the immunoprecipitation experiment. Aliquots of 50 μl of the Apaf-1 fraction, 100 μl of the Apaf-3 fraction, 0.5 μl (10 mg/ml) of purified cytochrome c, and dATP to a final concentration of 1 mM were added as indicated to a final volume of 500 μl of buffer A. 500 μg of BSA was also added to all reactions to reduce nonspecific binding to the beads. After incubating at 30°C for 20 min, aliquots (25 μl) of anti-caspase-9 antibody/protein A beads prepared as in Experimental Procedures were added to the reaction. After incubation with rotation overnight at 4°C, the beads were centrifuged and washed with 1 ml of buffer A three times. The beads were then resuspended in 60 μl of 1× SDS loading buffer. After boiling for 5 min, the beads were pelleted by centrifugation and the supernatants were collected. Aliquots of 15 μl of resulting supernatants were subjected to 8% SDS-PAGE followed by electroblotting to a nitrocellulose filter. Purified Apaf-1 (5 ng) was loaded on lane 13. The filter was probed with the serum against Apaf-1 (1:2000 dilution) as described in Zou et al. 1997. The antigen/antibody complexes were visualized by an ECL method as described in Experimental Procedures. The filter was exposed to Kodak X-OMAT X-ray film for 1 min at room temparature. P, preimmune serum, I immune serum. (B) Aliquots of 120 μl (900 μg protein) of the Hela cell S-100 extract immunodepleted of cytochrome c (see Figure 3) were incubated in the absence (lanes 1, 2, 5, and 6) or presence (lanes 3, 4, 7, and 8) of 1 mM dATP at 30°C for 20 min in a final volume of 500 μl of buffer A. Purified cytochrome c (5 μg) was added to lanes 3, 4, 7, and 8. Purified Apaf-1 (5 ng) was loaded on lane 9. After incubation, the samples were subjected to immunoprecipitation and Western blot analysis as described in (A). The filter was exposed to Kodak X-OMAT film for 1 s (lanes 1–8) or 15 s (lane 9).
（A）Apaf-1 和 Apaf-3 按照邹等人 1997 年的描述通过羟基磷灰石柱分离，并用于免疫沉淀实验。将 50μl 的 Apaf-1 分数，100μl 的 Apaf-3 分数，0.5μl（10mg/ml）的纯化细胞色素 c 和最终浓度为 1mM 的 dATP 加入到 500μl 缓冲液 A 中。500μg 的 BSA 也添加到所有反应中，以减少对珠子的非特异性结合。在 30°C 孵育 20 分钟后，将制备如实验程序中所述的 25μl 抗 caspase-9 抗体/蛋白 A 珠子的等分加入到反应中。在 4°C 旋转孵育过夜后，珠子被离心并用 1ml 缓冲液 A 洗涤三次。然后将珠子重悬于 60μl 的 1×SDS 加载缓冲液中。煮沸 5 分钟后，通过离心将珠子沉淀，收集上清液。取 15μl 的上清液进行 8% SDS-PAGE，然后进行电泳转印到硝酸纤维滤纸上。在第 13 条道上加载纯化的 Apaf-1（5ng）。如邹等人 1997 年所述，用针对 Apaf-1 的血清（1:2000 稀释度）探测滤纸。将抗原/抗体复合物按照实验程序中所述的 ECL 方法可视化。将滤纸暴露在室温下的 Kodak X-OMAT X 射线胶片上 1 分钟。P，未免疫血清，I 免疫血清。（B）取 120μl（900μg 蛋白质）的 Hela 细胞 S-100 提取物去除细胞色素 c（见图 3），在 500μl 缓冲液 A 的最终体积中，在 30°C 孵育 20 分钟，无 dATP 存在（1、2、5 和 6 道）或存在 1mM dATP（3、4、7 和 8 道）。将纯化的细胞色素 c（5μg）加入到 3、4、7 和 8 道。纯化的 Apaf-1（5ng）加载到第 9 道。孵育后，样品按照（A）中描述的进行免疫沉淀和 Western 印迹分析。将滤纸暴露在 Kodak X-OMAT 胶片上 1 秒（1-8 道）或 15 秒（第 9 道）。

To further demonstrate that the interaction of caspase-9 with Apaf-1 requires cytochrome c and dATP, we performed the same coimmunoprecipitation experiment with HeLa S-100 immunodepleted of cytochrome c (Figure 6B). This extract possessed no caspase-3 activating activity unless purified cytochrome c was added to the reaction (Figure 3, lanes 7–10 ). Apaf-1 was coimmunoprecipitated with caspase-9 in this extract only when both cytochrome c and dATP were added (lane 8). Preimmune serum did not precipitate Apaf-1 (lane 7).
为了进一步证明 caspase-9 与 Apaf-1 的相互作用需要细胞色素 c 和 dATP，我们对去除了细胞色素 c 的 HeLa S-100 免疫沉淀物进行了相同的共免疫沉淀实验（图 6B）。除非向反应中加入纯化的细胞色素 c，否则该提取物不具有 caspase-3 激活活性（图 3，第 7-10 条）。只有在同时加入细胞色素 c 和 dATP 时，Apaf-1 才会与该提取物中的 caspase-9 共同免疫沉淀（第 8 条）。未经免疫的血清不会沉淀 Apaf-1（第 7 条）。

Caspase-9 Binds to Apaf-1 through Interaction of CARDs
Caspase-9 通过 CARDs 的相互作用与 Apaf-1 结合

Several caspases, including caspase-9, have long prodomains at their NH₂ termini. This domain has been proposed to function as a CARD, allowing proteins with such domains to interact with each other (Hofmann et al. 1997). Although Apaf-1 is not a caspase, its NH₂-terminal region contains a CARD, suggesting that Apaf-1 may recruit caspase-9 through their respective CARDs (Zou et al. 1997). To test this possibility directly, we expressed the first 97 amino acids of Apaf-1 fused to a GST protein and studied its interaction with caspase-9. As shown in Figure 7, wild-type caspase-9 translated in vitro bound to the CARD of Apaf-1. The active site mutation, cysteine 287 to alanine, also bound to this fusion protein. Caspase-9 without its prodomain no longer bound. Caspase-3, which does not have a long prodomain, did not bind to the CARD of Apaf-1 either. GST alone did not bind caspase-9. In contrast to the results with full-length Apaf-1, we found that the CARD-GST fusion protein bound caspase-9 in the absence of cytochrome c and dATP (data not shown). These data suggest that, within the context of full-length Apaf-1, the CARD is not accessible for caspase-9 binding. Cytochrome c and dATP may induce a conformational change in Apaf-1 that exposes its CARD.
几种半胱氨酸蛋白酶，包括半胱氨酸蛋白酶-9，在它们的氨基末端有长的前序结构域。这个结构域被提议作为一个 CARD，使具有这种结构域的蛋白质能够相互作用（Hofmann 等人，1997 年）。虽然 Apaf-1 不是一种半胱氨酸蛋白酶，但其氨基末端区域含有一个 CARD，这表明 Apaf-1 可能通过它们各自的 CARD 招募半胱氨酸蛋白酶-9（Zou 等人，1997 年）。为了直接测试这种可能性，我们表达了 Apaf-1 的前 97 个氨基酸与 GST 蛋白融合，并研究了它与半胱氨酸蛋白酶-9 的相互作用。如图 7 所示，在体外翻译的野生型半胱氨酸蛋白酶-9 与 Apaf-1 的 CARD 结合。活性位点突变，半胱氨酸 287 至丙氨酸，也结合到这个融合蛋白上。没有其前序结构域的半胱氨酸蛋白酶-9 不再结合。没有长前序结构域的半胱氨酸蛋白酶-3 也不结合 Apaf-1 的 CARD。单独的 GST 不结合半胱氨酸蛋白酶-9。与全长 Apaf-1 的结果相反，我们发现 CARD-GST 融合蛋白在没有细胞色素 c 和 dATP 的情况下结合半胱氨酸蛋白酶-9（数据未显示）。这些数据表明，在全长 Apaf-1 的背景下，CARD 对半胱氨酸蛋白酶-9 的结合不可及。细胞色素 c 和 dATP 可能诱导 Apaf-1 发生构象变化，暴露其 CARD。

1. Download : Download high-res image (203KB)
   下载：下载高分辨率图像（203KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 7. Apaf-1 and Caspase-9 Interact via their CARDs
图 7. Apaf-1 和 Caspase-9 通过它们的 CARDs 相互作用

The CARD domain of Apaf-1 (residues 1–97) was subcloned into the bacterial expression vector pGEX-5X-3 in-frame with an N-terminal GST tag. GST and GST-APAF-1-CARD fusion protein were expressed in E. coli DH5α bacteria and then immobilized on glutathione-Sepharose. Labeled interacting proteins were prepared as in Figure 4, and aliquots of 1/10 of the input for binding assay were subjected directly to SDS-PAGE and visualilzed by autoradiography. Equal amounts of the indicated translation reactions were diluted to 150 μl with GST binding buffer (50 mM Tris-HCl [pH 7.6], 120 mM NaCl, and 0.5% Brij supplemented with protease inhibitors) and allowed to incubate with the immobilized GST or GST-Apaf-1(1–97) at 4°C for 2 hr with rotation. The beads were pelleted by centrifugation and washed four times with the same buffer and boiled for 3 min in 1× SDS sample buffer. After a brief centrifugation, the resulting supernatants were subjected to a 10% SDS gel and visualized by autoradiography.
Apaf-1 的 CARD 结构域（残基 1-97）被亚克隆到细菌表达载体 pGEX-5X-3 中，与 N-末端的 GST 标签相框架。 GST 和 GST-APAF-1-CARD 融合蛋白在大肠杆菌 DH5α细菌中表达，然后固定在谷胱甘肽-Sepharose 上。 标记的相互作用蛋白如图 4 中所述制备，并且绑定测定的输入的 1/10 的等分直接经过 SDS-PAGE 处理，并通过放射自显影进行可视化。 所示的翻译反应的相等量被稀释到 150μl 的 GST 结合缓冲液（50mM Tris-HCl [pH 7.6]，120mM NaCl 和 0.5% Brij，加入蛋白酶抑制剂）中，并与固定的 GST 或 GST-Apaf-1(1-97)在 4°C 下旋转孵育 2 小时。 将珠子通过离心沉淀并用相同缓冲液洗涤四次，然后在 1×SDS 样品缓冲液中煮沸 3 分钟。 经过简短的离心后，得到的上清液经过 10% SDS 凝胶处理，并通过放射自显影进行可视化。

Active Site Mutation of Caspase-9 Blocks Caspase-3 Activation and Apoptosis In Vivo
Caspase-9 的活性位点突变阻断了 Caspase-3 的激活和体内凋亡

Inasmuch as the active site mutant version of caspase-9 binds to Apaf-1, we designed experiments to determine whether this mutant protein exerts a dominant-negative influence on activation of caspase-3 and cellular apoptosis response in vivo.
鉴于 caspase-9 的活性位点突变体与 Apaf-1 结合，我们设计了实验来确定这种突变蛋白是否对 caspase-3 的激活和细胞凋亡反应在体内产生显性负影响。

When human breast carcinoma MCF-7 cells were transiently transfected with an expression vector, about 17% of the cells showed spontaneous apoptosis (Figure 8A). When the vector encoded full-length Apaf-1 or wild-type caspase-9, there was a slight increase to 26% and 29%, respectively. When Apaf-1 and wild-type caspase-9 were coexpressed, almost 60% of the cells underwent apoptosis. When the active site mutant of caspase-9 (C287A) was coexpressed with Apaf-1, the number of cells undergoing apoptosis decreased to about 10%.
当人类乳腺癌 MCF-7 细胞瞬时转染表达载体时，约 17%的细胞表现出自发性凋亡（图 8A）。当载体编码全长 Apaf-1 或野生型 caspase-9 时，分别增加到 26%和 29%。当 Apaf-1 和野生型 caspase-9 共同表达时，将近 60%的细胞发生凋亡。当 caspase-9 的活性位点突变体（C287A）与 Apaf-1 共同表达时，发生凋亡的细胞数量减少到约 10%。

1. Download : Download high-res image (161KB)
   下载：下载高分辨率图像（161KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 8. Inhibition of Apoptosis and Caspase-3 Activation by an Active Site Mutant of Caspase-9
图 8。通过 Caspase-9 的活性位点突变体抑制凋亡和 Caspase-3 激活

Human MCF-7 and 293 cells were transiently transfected with the indicated plasmids as described in the Experimental Procedures.
人类 MCF-7 和 293 细胞被瞬时转染，转染使用的质粒如实验程序中所述。

(A) The graph shows the percentage of round blue apoptotic MCF-7 cells as a function of total blue cells under each condition.
（A）图显示了在每种条件下圆形蓝色凋亡 MCF-7 细胞占总蓝色细胞的百分比。

(B) Human embryonic kidney cells (293 cells) were transfected with the indicated amount of Bax in pCIneo vector together with the indicated amount of plasmid containing C287A mutant caspase-9 as described in the Experimental Procedures. Cells were collected 30 hr after transfection, and the cell lysates were analyzed by Western blot analysis using a polyclonal antibody against human caspase-3.
（B）人类胚胎肾细胞（293 细胞）被转染与 pCIneo 载体中指定量的 Bax，以及包含 C287A 突变型 caspase-9 质粒的指定量，如实验程序所述。转染后 30 小时收集细胞，并使用针对人类 caspase-3 的多克隆抗体进行 Western blot 分析细胞裂解液。

Bax is a member of the bcl-2 family that opposes the action of bcl-2 and accelerates cell death when overexpressed in mammalian cells (Oltvai et al. 1993). Overexpression of Bax in human MCF-7 cells elicited apoptosis in nearly 90% of the transfected cells (Figure 8). Cotransfection with the active site mutant of caspase-9 reduced the percentage of cells undergoing apoptosis to about 20%, which is near the background level.
Bax 是 bcl-2 家族的成员，当在哺乳动物细胞中过度表达时，与 bcl-2 的作用相对立并加速细胞死亡（Oltvai 等，1993 年）。在人类 MCF-7 细胞中过表达 Bax 引发了近 90%的转染细胞凋亡（图 8）。与 caspase-9 的活性位点突变体共转染将正在凋亡的细胞百分比降低到约 20%，接近背景水平。

To confirm that the active site mutant of caspase-9 (C287A) blocks apoptosis by inhibiting caspase-3 activation, we analyzed caspase-3 in human embryonic kidney cells (293 cells) that were transfected with Bax, or Bax plus the mutant caspase-9 (C287A). As shown in Figure 8B, activation of caspase-3 was detected in cells that were overexpressing Bax (lane 2). The activation of caspase-3 was decreased to about 50% when an equal amount of mutant caspase-9 was coexpressed with Bax, and the activation of caspase-3 was completely blocked when a 4-fold excess amount of mutant caspase-9 was used (lanes 3–4).
为了确认半胱氨酸蛋白酶-9 的活性位点突变体（C287A）通过抑制半胱氨酸蛋白酶-3 的激活来阻止细胞凋亡，我们分析了转染了 Bax 或 Bax 加上突变型半胱氨酸蛋白酶-9（C287A）的人类胚胎肾细胞（293 细胞）中的半胱氨酸蛋白酶-3。如图 8B 所示，检测到了过表达 Bax 的细胞中半胱氨酸蛋白酶-3 的激活（第 2 条带）。当与 Bax 共同表达等量的突变型半胱氨酸蛋白酶-9 时，半胱氨酸蛋白酶-3 的激活减少约 50％，当使用 4 倍过量的突变型半胱氨酸蛋白酶-9 时，半胱氨酸蛋白酶-3 的激活完全被阻断（第 3-4 条带）。

Role of Apaf-1 Apaf-1 的作用

The current data suggest that Apaf-1 serves as a docking protein for caspase-9 and cytochrome c. Apaf-1 is a 130 kDa protein that possesses three distinct domains (Zou et al. 1997). The NH₂-terminal 85 amino acids are homologous to the CARD segment in the prodomain of several caspases, including CED-3, caspase-1, caspase-2, and caspase-9 (48, 61, 54, 7, 43). CARDs have been proposed to mediate the recruitment of caspases (Hofmann et al. 1997). Indeed, the CARD of Apaf-1 readily binds to caspase-9 (Figure 7). These data do not exclude the possibility that caspase-9 interacts with other regions of Apaf-1 as well. Apaf-1 binds caspase-9 only in the presence of cytochrome c and dATP. This suggests that the CARD of Apaf-1 is normally not exposed when cytochrome c is absent from the cytosol.
当前数据表明 Apaf-1 作为 caspase-9 和细胞色素 c 的对接蛋白。Apaf-1 是一种 130 kDa 的蛋白质，具有三个不同的结构域（Zou 等，1997 年）。N 端的 85 个氨基酸与几种 caspase 的前体中的 CARD 段同源，包括 CED-3、caspase-1、caspase-2 和 caspase-9（48、61、54、7、43）。已经提出 CARD 可能介导 caspase 的招募（Hofmann 等，1997 年）。事实上，Apaf-1 的 CARD 能够与 caspase-9 结合（图 7）。这些数据并不排除 caspase-9 与 Apaf-1 的其他区域也发生相互作用的可能性。Apaf-1 只在存在细胞色素 c 和 dATP 时与 caspase-9 结合。这表明当细胞色素 c 不在细胞质中时，Apaf-1 的 CARD 通常不暴露。

Role of Cytochrome c 细胞色素 c 的作用

Cytochrome c is absolutely required in our in vitro system for the activation of caspase-3 and caspase-9. When cytochrome c was depleted from HeLa cell S-100 extracts by a monoclonal antibody, the ability to activate caspases was abolished. Activity was restored when purified cytochrome c was added back (Figure 3). The essential role of cytochrome c was further demonstrated in the coimmunoprecipitation experiment. When cytochrome c was depleted, caspase-9 failed to bind Apaf-1 even in the presence of dATP. The binding was restored when purified cytochrome c was added (Figure 6). These data strongly support the suggestion that cytochrome c initiates apoptosis by inducing the formation of the Apaf-1/caspase-9 complex.
在我们的体外系统中，细胞色素 c 对于激活半胱天冬氨酸蛋白酶-3 和半胱天冬氨酸蛋白酶-9 是绝对必需的。当通过单克隆抗体从 HeLa 细胞 S-100 提取物中去除细胞色素 c 时，激活半胱天冬氨酸蛋白酶的能力被废除。当加回纯化的细胞色素 c 时，活性得以恢复（图 3）。细胞色素 c 的关键作用在共免疫沉淀实验中进一步得到证实。当细胞色素 c 被去除时，即使存在 dATP，半胱天冬氨酸蛋白酶-9 也无法结合 Apaf-1。当加入纯化的细胞色素 c 时，结合得以恢复（图 6）。这些数据强烈支持细胞色素 c 通过诱导 Apaf-1/半胱天冬氨酸蛋白酶-9 复合物的形成来启动凋亡的建议。

Holocytochrome c is normally present exclusively in mitochondria. Apocytochrome c that is newly synthesized in the cytosol cannot activate apoptosis (Yang et al. 1997). In response to a variety of apoptosis-inducing agents, cytochrome c is released from mitochondria to the cytosol (Liu et al., 1996; 18, 19, 20, 58). In cells overexpressing bcl-2, or its close family member bcl-xl, the release of cytochrome c is blocked, aborting the apoptotic response (18, 19, 20, 58). Cytochrome c binds to Apaf-1 in the absence of dATP (Zou et al. 1997), but this complex will not bind caspase-9 unless dATP or ATP is present (Figure 6). It is likely that the nucleotide induces a conformational change in the Apaf-1/cytochrome c complex that exposes the CARD domain of Apaf-1, allowing caspase-9 to bind. The precise region of Apaf-1 that binds cytochrome c has not yet been mapped.

Role of dATP/ATP

On the COOH-terminal side of the CARD domain, Apaf-1 contains a region of 320 amino acids that is homologous to CED-4. This region contains Walker's A and B boxes and the nucleotide p-loop (52, 62), suggesting that it serves as a nucleotide binding site. Mutations of residues in CED-4 that are required for nucleotide binding destroy its ability to promote caspase activation (5, 36). Similar mutations of Apaf-1 also obliterate its activity (S. M. S. and E. S. A., unpublished data). ATP will replace dATP, but the required concentration is much higher (1 μM for dATP versus 1 mM for ATP) (Figure 5). The intracellular concentrations of ATP and dATP are in the range of 0.2–10 mM and 10–20 μM, respectively (Skoog and Bjursell 1974), raising the possibility that ATP, as well as dATP, may support apoptosis within the cell. Our observation that nonhydrolyzable analogs of ATP fail to subsitute for ATP suggests that hydrolysis of the high-energy bond is required for caspase activation.

The suggestion that ATP is required to carry out the cell suicide program is supported by recent observation that cells depleted of ATP undergo necrosis instead of apoptosis in response to apoptotic stimuli (8, 23). Restoring ATP levels restored the apoptotic response (8, 23).

The Role of Caspase-9

All of our data are consistent with the suggestion that caspase-9 is the caspase that is directly activated by Apaf-1 and cytochrome c, at least in the cellular extracts that we studied. When the HeLa S-100 extracts were immunodepleted of caspase-9, they lost the ability to activate caspase-3, and activity was restored by addition of purified caspase-9. The initial cleavage of caspase-9, whether it is through autocatalysis or another mechanism, is currently under investigation.

Several other caspases, including caspase-1, -2, -8, and -10, also contain long NH₂-terminal CARDs like that of caspase-9 (48, 54, 3, 29, 44). It is possible that one or more of these caspases might substitute for caspase-9 in tissues in which their expression is high.
其他几种半胱氨酸蛋白酶，包括半胱氨酸蛋白酶-1、-2、-8 和-10，也包含类似于半胱氨酸蛋白酶-9（48, 54, 3, 29, 44）的长 NH ₂ -末端 CARD。可能有一种或多种这些半胱氨酸蛋白酶在其表达量高的组织中可以替代半胱氨酸蛋白酶-9。

General Methods and Materials
一般方法和材料

We obtained dATP and other nucleotides from Pharmacia, radioactive materials from Amersham, and molecular weight standards for SDS-PAGE and gel filtration chromatography from Bio-Rad and GIBCO-BRL. Protein concentrations were determined by the Bradford method; general molecular biology methods were used as described in Sambrook et al. 1989.
我们从 Pharmacia 获得了 dATP 和其他核苷酸，从 Amersham 获得了放射性材料，从 Bio-Rad 和 GIBCO-BRL 获得了 SDS-PAGE 和凝胶过滤色谱的分子量标准。蛋白质浓度是通过 Bradford 方法确定的；一般的分子生物学方法如 Sambrook 等人 1989 年所述。

Assay for Caspase-3 Activation
Caspase-3 活化测定

Caspase-3 was translated and purified as described (Liu et al. 1996a). A 3 μl aliquot of the in vitro–translated caspase-3 was incubated with the indicated protein fraction in the presence of 1 mM dATP and 1 mM of additional MgCl₂ at 30°C for 1 hr in a final volume of 20 μl of buffer A (20 mM HEPES-KOH [pH 7.5], 10 mM KCl, 1.5 mM MgCl₂, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, and 0.1 mM PMSF). At the end of the incubation, 7 μl of 4× SDS sample buffer was added to each reaction. After boiling for 3 min, each sample was subjected to a 15% SDS-PAGE. The gel was transferred to a nitrocellulose filter, which was subsequently exposed to a phosphorimaging plate and visualized in a Fuji BAS-1000 Phosphorimager.
Caspase-3 被翻译并纯化，如前所述（Liu 等，1996a）。在 20μl 缓冲液 A（20mM HEPES-KOH [pH 7.5]，10mM KCl，1.5mM MgCl ₂ ，1mM EDTA 钠，1mM EGTA 钠，1mM 二硫苏糖醇，和 0.1mM PMSF）中，将 3μl 体外翻译的 caspase-3 与所示蛋白质分数一起在 30°C 下与 1mM dATP 和 1mM 额外 MgCl ₂ 孵育 1 小时。孵育结束时，向每个反应中加入 7μl 的 4×SDS 样品缓冲液。沸腾 3 分钟后，每个样品均经过 15% SDS-PAGE。凝胶转移到硝酸纤维滤纸上，随后暴露于磷光成像板并在 Fuji BAS-1000 磷光成像仪中可视化。

Purification of Apaf-3 from HeLa S-100
从 HeLa S-100 纯化 Apaf-3

All purification steps were carried out at 4°C. All chromatography steps except the SP-Sepharose column (Pharmacia), hydroxylapatite column (Bio-Rad), and Q-Sepharose (Pharmacia) were carried out using an automatic fast protein liquid chromatography (FPLC) station (Pharmacia).
所有纯化步骤均在 4°C 下进行。除 SP-Sepharose 柱（Pharmacia）、羟基磷灰石柱（Bio-Rad）和 Q-Sepharose（Pharmacia）外，所有色谱步骤均使用自动快速蛋白液相色谱（FPLC）站（Pharmacia）进行。

HeLa S-100 (700 ml [4.9 g of protein]) from 100 liters of suspension-cultured HeLa cells was prepared as described in Liu et al. 1996b and applied to a SP-Sepharose column (200 ml bed vol) equilibrated with buffer A. The 800 ml flow-through fraction (3648 mg of protein) was collected and loaded directly onto a hydroxylapaptite column (50 ml bed vol) equilibrated with buffer A. Flow-through fraction (2741 mg of protein) of the hydroxylapatite column was directly loaded on to a Q-Sepharose column (100 ml bed vol). The column was washed with three column vol of buffer A. The bound material was eluted with three 200 ml steps elution with buffer A containing 0.1 M NaCl, 0.2 M NaCl, and 0.3 M NaCl. The fractions from step elution were assayed for Apaf-3 activity. Ammonium sulfate (1 M) was added directly to the active faction (0.2 M NaCl elution, 279 mg of protein), and after rotating at 4°C for 1 hr, the precipitate was pelleted by centrifugation in a SA-600 rotor (Sorvall) at 15,000 rpm for 20 min, and the supernatant was directly loaded onto a Phenyl-Superose 10/10 column (Pharmacia) equilibrated with buffer A containing 1 M ammonium sulfate. The column was eluted with a 200 ml linear gradient of buffer A containing 1 M ammonium sulfate to buffer A. Fractions of 4 ml were collected and assayed for Apaf-3 activity after diluting 1/10 with buffer A. Active fractions (20 ml, 9.8 mg of protein) were pooled and dialyzed against buffer A and then loaded onto a 5 ml heparin-agarose column (Pharmacia) equilibrated with buffer A. The column was washed with 20 ml of buffer A containing 100 mM NaCl and eluted with a 50 ml linear gradient from 100–300 mM NaCl, both in buffer A. Fractions of 4 ml were collected and assayed for Apaf-3 activity. The active fractions (12 ml, 3.4 mg of protein) were pooled and loaded directly onto a Superdex 200 16/60 gel-filtration column equilibrated with buffer A containing 100 mM NaCl. The column was eluted with the same buffer (6 runs), and fractions of 4 ml were collected starting from 30 ml of elution. The fractions were assayed for Apaf-3 activity and the active fractions were pooled (48 ml, 480 μg of protein). After diluting with equal volume of buffer A, the sample was loaded directly onto a Mono Q 5/5 column (Pharmacia) equilibrated with buffer A containing 50 mM NaCl. The column was eluted with a 20 ml linear gradient from 50–250 mM NaCl, both in buffer A. Fractions of 1 ml were collected and assayed for Apaf-3 activity. A 0.5 ml aliquot of the peak Apaf-3 fraction (12 μg) from the Mono Q column was directly loaded onto a 5 ml 10%–30% linear glycerol gradient in buffer A. After centrifugation at 55,000 rmp for 16 hr in a SW 60 rotor (Beckman), fractions of 0.5 ml were collected from bottom to top and assayed for Apaf-3 activity.
HeLa S-100（700 毫升[4.9 克蛋白质]）从 100 升悬浮培养的 HeLa 细胞中制备，方法如刘等人 1996b 所述，并应用于预先用缓冲液 A 平衡的 SP-Sepharose 柱（200 毫升床体积）。收集 800 毫升流过液（3648 毫克蛋白质），直接加载到预先用缓冲液 A 平衡的羟基磷灰石柱（50 毫升床体积）。羟基磷灰石柱的流过液（2741 毫克蛋白质）直接加载到预先用缓冲液 A 平衡的 Q-Sepharose 柱（100 毫升床体积）。柱子用三倍缓冲液 A 洗涤。结合物质用含 0.1M NaCl、0.2M NaCl 和 0.3M NaCl 的缓冲液 A 进行三次 200 毫升梯度洗脱。梯度洗脱的分数进行 Apaf-3 活性测定。将硫酸铵（1M）直接加入活性分离物（0.2M NaCl 洗脱，279 毫克蛋白质），在 4°C 旋转 1 小时后，通过在 SA-600 转子（Sorvall）中以 15000 转/分钟离心 20 分钟，将沉淀物沉淀，上清液直接加载到预先用含 1M 硫酸铵的缓冲液 A 平衡的 Phenyl-Superose 10/10 柱（Pharmacia）。柱子用含 1M 硫酸铵的缓冲液 A 进行 200 毫升线性梯度洗脱至缓冲液 A。收集 4 毫升分数，并在稀释 1/10 的缓冲液 A 中进行 Apaf-3 活性测定。活性分离物（20 毫升，9.8 毫克蛋白质）混合并对缓冲液 A 进行透析，然后加载到预先用缓冲液 A 平衡的 5 毫升肝素琼脂糖柱（Pharmacia）。柱子用含 100mM NaCl 的缓冲液 A 进行 20 毫升洗脱，并用从 100-300mM NaCl 的 50 毫升线性梯度洗脱，两者均在缓冲液 A 中。收集 4 毫升分数，并进行 Apaf-3 活性测定。活性分离物（12 毫升，3.4 毫克蛋白质）混合并直接加载到预先用含 100mM NaCl 的缓冲液 A 平衡的 Superdex 200 16/60 凝胶过滤柱。 该柱用相同缓冲液洗脱（6 次），并从 30 毫升洗脱开始收集 4 毫升的分级。对 Apaf-3 活性进行测定，将活性分级混合（48 毫升，480 微克蛋白）。将样品与等体积的缓冲液 A 稀释后，直接加载到含有 50 毫摩尔氯化钠的缓冲液 A 平衡的 Mono Q 5/5 柱（Pharmacia）。该柱用 50-250 毫摩尔氯化钠的 20 毫升线性梯度（均在缓冲液 A 中）洗脱。收集 1 毫升的分级并对 Apaf-3 活性进行测定。从 Mono Q 柱的峰值 Apaf-3 分级（12 微克）中直接加载到缓冲液 A 中 10%-30%线性甘油梯度的 5 毫升中。在 SW 60 转子（Beckman）中以 55,000 转/分钟的速度离心 16 小时后，从底部到顶部收集 0.5 毫升的分级并对 Apaf-3 活性进行测定。

Production of Caspase-9 Fusion Protein
Caspase-9 融合蛋白的生产

The full-length caspase-9 coding region (Srinivasula et al. 1996a) was cloned into the BamHI site of pET-15b vector (Novagen). The expression plasmid was transformed into bacteria BL21 cells (Novagen). In a typical caspase-9 preparation, a 5 ml overnight culture of bacteria containing the caspase-9 expression vector was added into a 1 liter LB broth, cultured for 3 hr by shaking at 220 rpm in 37°C. Isopropyl-1-thio-B-D-galactopyranoside (IPTG) was added to the culture in a final concentration of 1 mM and continued shaking for another 2 hr. The bacteria were pelleted by centrifugation, and the bacterial pellet was resuspended in 10 vol of buffer A. The resuspended cells were lysed in buffer A by sonication. After centrifugation at 10,000 g for 15 min, the supernatant was loaded onto a nickel affinity column (2 ml). The column was washed with 20 ml buffer A followed by 20 ml buffer A containing 1 M NaCl. After reequilibrating the column with 20 ml buffer A, the bound protein was eluted with buffer A containing 250 mM imidazole. The identity of caspase-9 was verified by SDS-PAGE followed by Coomassie blue staining and used to immunize rabbits.
将全长 caspase-9 编码区（Srinivasula 等人，1996a 年）克隆到 pET-15b 载体（Novagen）的 BamHI 位点中。表达质粒被转化到细菌 BL21 细胞（Novagen）中。在典型的 caspase-9 制备中，含有 caspase-9 表达载体的 5 毫升过夜培养细菌被加入到 1 升 LB 培养基中，在 37°C 下以 220 rpm 振荡培养 3 小时。将异丙基-1-硫代-B-D-半乳糖苷（IPTG）加入到培养基中，最终浓度为 1 毫摩尔，并继续振荡培养 2 小时。通过离心将细菌沉淀，再用 10 倍体积的 A 缓冲液重悬细胞。用声波破碎法在 A 缓冲液中裂解细胞。在 10,000 g 的离心后，上清液被加载到镍亲和柱（2 毫升）上。用 20 毫升 A 缓冲液洗涤柱，然后用含 1 M NaCl 的 20 毫升 A 缓冲液洗涤。重新用 20 毫升 A 缓冲液平衡柱后，用含 250 毫摩尔咪唑的 A 缓冲液洗脱结合的蛋白质。通过 SDS-PAGE 和考马斯亮蓝染色验证 caspase-9 的身份，并用于免疫兔子。

The active caspase-9 was generated by transforming bacterial competent cells BL21(DE3) (Novagen) with a full-length caspase-9, an independent clone that differs from the published sequence at amino acid 197, P197L (Srinivasula et al. 1996a), cloned into pET-21b vector (Novagen). The expression of the recombinant caspase-9 was induced with IPTG for 3 hr at room temparature. The recombinant protein was subsequently purified as described above.
通过将含有全长 caspase-9 的独立克隆体（与已发表的序列在氨基酸 197 位点 P197L 处不同）克隆到 pET-21b 载体（Novagen）中，转化细菌 BL21（DE3）（Novagen）的活性 caspase-9 被产生。重组 caspase-9 的表达在室温下用 IPTG 诱导 3 小时。随后按上述方法纯化重组蛋白质。

Western Blot Analysis 西方印迹分析

Western blot analysis for Apaf-1 was performed as described previously (Zou et al. 1997). Anti-caspase-9 antiserum was generated by immunizing rabbits with a recombinant caspase-9 fusion protein (see above). Immunoblot analysis was performed with a horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G using enhanced chemiluminescence Western blotting detection reagents (Amersham).

Immunodepletion of Caspase-9 from HeLa S-100
从 HeLa S-100 中免疫去除 Caspase-9

An aliquot of 500 μl of protein A–agarose (Santa Cruz) was incubated with an aliquot of 500 μl of preimmune or immune anti-caspase-9 serum at 4°C overnight. The antibody/protein A–agarose beads were then pelleted by centrifugation and washed five times with buffer A. The beads were resuspended in 1 ml of buffer A.
取 500μl 蛋白 A 琼脂（Santa Cruz）与 500μl 预免疫或免疫抗 caspase-9 血清在 4°C 孵育过夜。然后，通过离心将抗体/蛋白 A 琼脂珠沉淀并用缓冲液 A 洗涤五次。将珠子重悬于 1ml 缓冲液 A 中。

An aliquot of 50 μl of these beads was incubated with 100 μl BSA dissolved in buffer A (50 mg/ml) at room temperature for 2 hr. The beads were pelleted by centrifugation and incubated overnight at 4°C with 1 ml (8.3 mg/ml protein) HeLa cell S-100 extracts. The beads were subsequently pelleted by centrifugation, and the supernatant was used as extracts immunodepleted of caspase-9.
这些珠子的 50 微升等分子与 100 微升在缓冲液 A 中溶解的 BSA 在室温下孵育 2 小时。珠子通过离心沉淀，并在 4°C 孵育过夜与 1 毫升（8.3 毫克/毫升蛋白质）HeLa 细胞 S-100 提取物。珠子随后通过离心沉淀，上清液被用作去除 caspase-9 的提取物。

Transfection of MCF-7 and 293 Cells
MCF-7 和 293 细胞的转染

Human MCF-7 cells were set at 1 × 10⁵ cells per well in a 12-well plate at day 0. On day 2, the cells were transfected with 0.5 μg of pRSC-LacZ double expression vector (Invitrogen) plus 0.5 μg of pFLAG-CMV-2 vector (IBI Kodak) (well 1); or 0.5 μg of pRSC-LacZ vector plus 0.5 μg of pFlag-CMV-2 containing full-length Apaf-1 (pApaf-1/flag) (well 3); or 0.5 μg of pRSC-LacZ vector containing wild-type caspase-9 (pCasp-9) plus 0.5 μg of pFLAG-CMV-2 vector (well 2); or 0.5 μg of pRSC-LacZ vector containing the wild-type caspase-9 plus 0.5 μg of pFLAG-CMV-2 vector containing full-length Apaf-1 (well 4); or 2 μg of pCasp-9/C287A plus 0.5 μg of pApaf-1/flag and 0.5 μg of pRSC-LacZ vector (well 5); or 2 μg of pcDNA 3 vector plus 0.5 μg of full-length Bax in pRSC-LacZ (pRSC-LacZ-Bax) (well 6); or 2 μg of pCasp-9/C287A plus 0.5 μg of pRSC-LacZ-Bax (well 7) using the LipofectAMINE (Life Technologies, Inc.) method. The amount of DNA in all transfection experiments was made equal by including respective amounts of vector. Cells were stained for β-galactosidase expression and examined for morphological signs of apoptosis 30 hr after transfection.
人类 MCF-7 细胞在第 0 天以每孔 1 × 10 ⁵ 个细胞的数量置于 12 孔板中。第 2 天，细胞使用 0.5 μg pRSC-LacZ 双表达载体（Invitrogen）加上 0.5 μg pFLAG-CMV-2 载体（IBI Kodak）进行转染（孔 1）；或者使用 0.5 μg pRSC-LacZ 载体加上 0.5 μg 含全长 Apaf-1 的 pFlag-CMV-2 载体（孔 3）；或者使用含野生型 caspase-9 的 0.5 μg pRSC-LacZ 载体加上 0.5 μg pFLAG-CMV-2 载体（孔 2）；或者使用含野生型 caspase-9 的 0.5 μg pRSC-LacZ 载体加上含全长 Apaf-1 的 0.5 μg pFLAG-CMV-2 载体（孔 4）；或者使用 2 μg pCasp-9/C287A 加上 0.5 μg pApaf-1/flag 和 0.5 μg pRSC-LacZ 载体（孔 5）；或者使用 2 μg pcDNA 3 载体加上含全长 Bax 的 0.5 μg pRSC-LacZ（pRSC-LacZ-Bax）（孔 6）；或者使用 2 μg pCasp-9/C287A 加上 0.5 μg pRSC-LacZ-Bax（孔 7）使用 LipofectAMINE（Life Technologies, Inc.）方法进行转染。所有转染实验中 DNA 的量通过包含相应量的载体使之相等。细胞在转染后 30 小时染色检测β-半乳糖苷酶表达并观察凋亡的形态学特征。

Human embryonic kidney cells (293 cells) were set at 4 × 10⁵ per well in a 6-well plate at day 0. On day 2, the cells were transfected with 0.5 μg of Bax in pCIneo vector together with the indicated amount of plasmid containing C287A mutant caspase-9 as described above.
人类胚胎肾细胞（293 细胞）在第 0 天以每孔 4 × 10 ⁵ 个细胞的数量置于 6 孔板中。第 2 天，细胞使用 0.5 μg pCIneo 载体中的 Bax 与上述所述的含 C287A 突变 caspase-9 的质粒进行转染。