A novel form of DAP5 protein accumulates in apoptotic cells as a result of caspase cleavage and internal ribosome entry site-mediated translation
A novel form of DAP5 protein accumulates in apoptotic cells as a result of caspase cleavage and internal ribosome entry site-mediated translation
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DOI：
10.1128/mcb.20.2.496-506.2000
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发表时间：
2000-01-01
影响因子：
5.3
通讯作者：
Kimchi, A
中科院分区：
文献类型：
--
作者：
Henis-Korenblit, S;Strumpf, NL;Kimchi, A
死亡相关蛋白5（DAP5）（也称为p97和NAT1）是翻译起始因子4G（eIF4G）家族的成员，其缺乏eIF4E结合位点。先前基于该蛋白的一个显性负性片段可防止细胞死亡这一发现，它被认为与细胞凋亡有关。在此我们探讨它在细胞凋亡期间的功能以及在翻译和翻译后水平影响该蛋白的两种不同的调控层次。发现DAP5蛋白在790位的一个半胱天冬酶切割位点被切割，以响应活化的Fas或p53，产生一个86 kDa的C末端截短蛋白，该蛋白能够与eIF4A和eIF3形成复合物。有趣的是，尽管凋亡细胞中的总体翻译速率根据帽依赖翻译的两种主要介导因子eIF4GI和eIF4GII的同时降解而降低了60% - 70%，但DAP5蛋白的翻译速率却被选择性地维持。在DAP5 mRNA的5'非翻译区鉴定出一个内部核糖体进入位点（IRES）元件，当它被亚克隆到双顺反子载体中时能够指导报告基因的翻译。尽管在Fas诱导的细胞凋亡期间从这个转染载体进行的帽依赖翻译减少了，但通过DAP5 IRES的翻译却被选择性地维持。向无细胞体系中添加重组DAP5/p97或DAP5/p86优先增强了通过DAP5 IRES的翻译，而通过添加一个显性负性DAP5片段中和网织红细胞裂解物中的内源性DAP5则干扰了这种翻译。在这些功能测定中，DAP5/p86凋亡形式比DAP5/p97更有效。总之，数据表明DAP5是一种半胱天冬酶激活的翻译因子，它至少从其自身的IRES介导不依赖帽的翻译，从而产生一个正反馈回路，负责在细胞凋亡期间DAP5的持续翻译。
Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5' untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.