Saleh A, Srinivasula S M, Acharya S, Fishel R, Alnemri E S
Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1999 Jun 18;274(25):17941-5. doi: 10.1074/jbc.274.25.17941.
To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we produced recombinant full-length Apaf-1 and purified it to complete homogeneity. Here we show using gel filtration that full-length Apaf-1 exists as a monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochrome c in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochrome c-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5'-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex.
为阐明Apaf-1激活procaspase-9的机制，我们制备了重组全长Apaf-1并将其纯化至完全同质。在此我们通过凝胶过滤显示，全长Apaf-1以单体形式存在，在与细胞色素c和dATP结合后可转化为由至少八个亚基组成的寡聚复合物。Apaf-1在无dATP时可与细胞色素c结合，但不形成寡聚复合物。然而，当将dATP添加到与细胞色素c结合的Apaf-1复合物中时，会发生完全寡聚化，这表明寡聚化是由dATP的水解驱动的。这一观点得到了以下观察结果的支持：ATP可诱导Apaf-1 - 细胞色素c复合物的寡聚化，而非水解不可的腺苷5'-O-(硫代三磷酸)则不能。与缺乏WD-40结构域的自发寡聚化的Apaf-530一样，寡聚全长Apaf-1 - 细胞色素c复合物在无额外dATP或细胞色素c的情况下可结合并加工procaspase-9。然而，与截短的Apaf-530复合物不同，全长Apaf-1复合物在加工后可释放成熟的caspase-9。一旦释放，成熟的caspase-9可加工procaspase-3，启动caspase级联反应。这些观察结果表明，细胞色素c和dATP是Apaf-1寡聚化所必需的，并表明WD-40结构域在全长Apaf-1的寡聚化以及从Apaf-1寡聚复合物中释放成熟caspase-9方面发挥重要作用。
