Onuki Reiko, Nagasaki Akira, Kawasaki Hiroaki, Baba Tadashi, Uyeda Taro Q P, Taira Kazunari
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan.
Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):14716-21. doi: 10.1073/pnas.232177599. Epub 2002 Oct 30.
When cells are exposed to death-inducing molecules such as tumor necrosis factor-alpha or Fas, caspase 8 is activated and cleaves an apoptotic facilitator, Bid, that is a member of the Bcl-2 family. After additional modification, the C-terminal moiety of Bid is translocated to the mitochondria and induces the release of cytochrome c into the cytoplasm. In an attempt to directly observe the cleavage of Bid and the following events in living cells, we constructed a vector that encoded Bid fused with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) (YFP-Bid-CFP). On expression of YFP-Bid-CFP in mammalian cells, we were able to observe the efficient transfer of energy from excited CFP to YFP within the YFP-Bid-CFP molecule and, importantly, the fusion protein YFP-Bid-CFP was fully functional in cells. When YFP-Bid-CFP was cleaved by caspase 8, on activation by anti-Fas Abs but not by Abeta or tunicamycin, no such transfer of energy was detected. To our knowledge, this is the first report of (i) visualization of the activation of Bid by proteolytic cleavage, with direct observation of the cleavage of YFP-Bid-CFP in the cytoplasm and subsequent translocation of the cleaved Bid to mitochondria and (ii) the absence of Abeta- or tunicamycin-mediated significant activation of caspase 8 in individual living cells.
当细胞暴露于诸如肿瘤坏死因子-α或Fas等诱导死亡的分子时，半胱天冬酶8被激活并切割凋亡促进因子Bid，Bid是Bcl-2家族的成员。经过进一步修饰后，Bid的C末端部分易位至线粒体并诱导细胞色素c释放到细胞质中。为了直接观察Bid的切割以及活细胞中的后续事件，我们构建了一个编码与黄色荧光蛋白（YFP）和青色荧光蛋白（CFP）融合的Bid的载体（YFP-Bid-CFP）。在哺乳动物细胞中表达YFP-Bid-CFP时，我们能够观察到YFP-Bid-CFP分子内从激发态的CFP到YFP的有效能量转移，重要的是，融合蛋白YFP-Bid-CFP在细胞中具有完全功能。当YFP-Bid-CFP被半胱天冬酶8切割时，在抗Fas抗体激活而非Aβ或衣霉素激活时，未检测到这种能量转移。据我们所知，这是第一份关于（i）通过蛋白水解切割可视化Bid的激活，直接观察细胞质中YFP-Bid-CFP的切割以及切割后的Bid易位至线粒体，以及（ii）在单个活细胞中Aβ或衣霉素介导的半胱天冬酶8无明显激活的报告。
