Mitochondrial release of pro-apoptotic proteins -: Electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes*
Mitochondrial release of pro-apoptotic proteins -: Electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes*
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DOI：
10.1074/jbc.m411106200
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发表时间：
2005-01-21
影响因子：
4.8
通讯作者：
Kluck, RM
中科院分区：
文献类型：
--
作者：
Uren, RT;Dewson, G;Kluck, RM
细胞凋亡启动的关键步骤是从线粒体膜间隙释放细胞色素 c 和其他促凋亡蛋白，如 Smac/DIABLO、Omi/HtrA2、凋亡诱导因子 (AIF) 和核酸内切酶 G (EndoG)。然而，关于所有这些蛋白质是否在不同系统中释放，存在着差异。我们的结果表明，未能观察到细胞色素 c 的释放可能是由于使用了不同的缓冲液所致，因为经过 caspase-8 切割的人 Bid (tBid) 透化后，细胞色素 c 从线粒体解离高度依赖于离子强度，需要 50 - 80 mM KCl、NaCl 或 LiCl。此外，使用低离子强度缓冲液从凋亡细胞中分离的线粒体结合了更大比例的内源细胞色素c。与细胞色素 c 相比，Smac/DIABLO 和 Omi/HtrA2 的释放与离子强度无关，而 AIF 和 EndoG 的行为就好像它们暴露于膜间空间，但束缚在内膜上或内膜内。 AIF 和 EndoG 也不是由活性半胱天冬酶释放的，这表明它们对细胞凋亡的参与可能是有限的。总之，虽然 tBid 使外膜渗透到细胞色素 c、Smac/DIABLO 和 Omi/HtrA2，但细胞色素 c 在细胞凋亡过程中的释放将被低估，除非保持足够的离子强度以克服细胞色素 c 与膜的静电缔合。
A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50 - 80 mM KCl, NaCl, or LiCl. In addition, mitochondria isolated from apoptotic cells using low ionic strength buffer bound a greater proportion of endogenous cytochrome c. In contrast to cytochrome c, Smac/DIABLO and Omi/HtrA2 were released independent of ionic strength, and AIF and EndoG behaved as if they are exposed to the intermembrane space but tethered to or within the inner membrane. AIF and EndoG were also not released by active caspases, which suggests their involvement in apoptosis may be limited. In summary, whereas tBid permeabilizes the outer membrane to cytochrome c, Smac/DIABLO, and Omi/HtrA2, the release of cytochrome c during apoptosis will be underestimated unless sufficient ionic strength is maintained to overcome the electrostatic association of cytochrome c with membranes.