Komarov Andrei P, Rokhlin Oskar W, Yu Chang-An, Gudkov Andrei V
Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14453-8. doi: 10.1073/pnas.0807549105. Epub 2008 Sep 16.
Functional selection of genetic suppressor elements (GSEs), engineered gene fragments that interfere with the function of a particular gene product, was used to identify regulators of FAS-induced apoptosis. Chicken DF-1 cells expressing human FAS receptor and susceptible to FAS-induced apoptosis were infected with a GSE library consisting of randomly fragmented normalized chicken cDNAs in a replication-competent avian retroviral vector. Virus-producing cells were subjected to several rounds of selection using FAS agonistic antibodies, resulting in isolation of a set of GSEs conferring resistance to FAS-induced apoptosis. Surprisingly, one of the isolated GSEs encoded a 42 amino acid-long polypeptide derived from the C-terminal half of cytochrome b (Cyt b) encoded by the mitochondrial genome. Subsequent experiments showed that caspase 8-dependent cleavage of mitochondrial Cyt b and translocation of its C-terminal half into the cytoplasm occurred during FAS-induced apoptosis in both chicken and human cells. Ectopic cytoplasmic expression of either full-length Cyt b or its C-terminal half in several human cell lines induced apoptosis, which could be suppressed by the isolated GSE, but not by Bcl2 over-expression or Apaf-1 or cytochrome c knock-down. These results reveal a cytochrome c-independent branch of FAS-induced apoptosis involving cleavage and cytoplasmic release of mitochondrial Cyt b.
基因抑制元件（GSEs）是经改造的基因片段，可干扰特定基因产物的功能，利用其功能筛选来鉴定FAS诱导凋亡的调节因子。表达人FAS受体且对FAS诱导凋亡敏感的鸡DF-1细胞，被一种GSE文库感染，该文库由复制能力强的禽逆转录病毒载体中的随机片段化标准化鸡cDNA组成。产生病毒的细胞用FAS激动抗体进行多轮筛选，从而分离出一组赋予对FAS诱导凋亡抗性的GSEs。令人惊讶的是，其中一个分离出的GSE编码一种42个氨基酸长的多肽，该多肽来源于线粒体基因组编码的细胞色素b（Cyt b）的C端一半。随后的实验表明，在鸡和人细胞的FAS诱导凋亡过程中，线粒体Cyt b发生半胱天冬酶8依赖性切割，其C端一半转运到细胞质中。在几种人细胞系中，全长Cyt b或其C端一半的异位细胞质表达诱导凋亡，这种凋亡可被分离出的GSE抑制，但不能被Bcl2过表达或Apaf-1或细胞色素c敲低所抑制。这些结果揭示了FAS诱导凋亡的一条不依赖细胞色素c的分支，涉及线粒体Cyt b的切割和细胞质释放。
