Ubiquitin-mediated degradation of the proapoptotic active form of bid - A functional consequence on apoptosis induction
Ubiquitin-mediated degradation of the proapoptotic active form of bid - A functional consequence on apoptosis induction
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DOI：
10.1074/jbc.m001083200
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发表时间：
2000-07-14
影响因子：
4.8
通讯作者：
Dimmeler, S
中科院分区：
文献类型：
--
作者：
Breitschopf, K;Zeiher, AM;Dimmeler, S
在基础条件下，促凋亡蛋白Bid是长寿命蛋白。促凋亡刺激物如肿瘤坏死因子-α（TNF α）或Fas诱导其半胱天冬酶-8介导的裂解成两个片段。Bid（tBid）的COOH末端切割片段定位于线粒体膜并触发细胞色素c的释放。在这里，我们表明，tBid是泛素化，随后降解的26 S蛋白酶体。蛋白酶体抑制剂MG-132和lactacystin显著抑制tBid的降解。相反，半胱天冬酶特异性或溶酶体抑制剂不影响tBid稳定性。此外，tBid内推定的泛素受体位点的突变导致通过脉冲追踪分析评估的稳定蛋白质。为了解决tBid降解是否可以通过与其他Bcl-2样蛋白的相互作用来调节，进行了共转染研究。然而，既不存在促凋亡Bax也不存在抗凋亡Bcl-2或Bcl-XL影响tBid降解。最后，我们确定了tBid降解的功能作用。与野生型tBid相比，稳定的tBid蛋白的过表达显著增强细胞色素c释放和随后的凋亡诱导约2倍。同样，tBid诱导的细胞凋亡大大放大了抑制tBid降解蛋白酶体特异性抑制剂MG-132。因此，tBid的蛋白酶体降解限制了活细胞中凋亡的程度。
Under basal conditions, the proapoptotic protein Bid is a long-lived protein. Pro-apoptotic stimuli such as tumor necrosis factor-alpha (TNF alpha) or Fas induce its caspase-8-mediated cleavage into two fragments. The COOH-terminal cleavage fragment of Bid (tBid) becomes localized to mitochondrial membranes and triggers the release of cytochrome c. Here we show that tBid is ubiquitinated and subsequently degraded by the 26 S proteasome. Degradation of tBid is significantly inhibited by the proteasome inhibitors MG-132 and lactacystin. In contrast, caspase-specific or lysosomal inhibitors do not affect tBid stability. Furthermore, mutation of the putative ubiquitin acceptor sites within tBid results in a stabilized protein as assessed by pulse-chase analysis. To address whether tBid degradation might be regulated by interaction with other Bcl-2-like proteins, cotransfection studies were performed. However, neither the presence of proapoptotic Bax nor antiapoptotic Bcl-2 or Bcl-XL affected tBid degradation. Finally, we determined the functional role of tBid degradation. Overexpression of stabilized tBid proteins significantly enhanced cytochrome c release and subsequent apoptosis induction approximately 2-fold compared with wild type tBid. Similarly, tBid-induced apoptosis was considerably amplified by inhibition of tBid degradation using the proteasome-specific inhibitor MG-132. Thus, proteasomal degradation of tBid limits the extent of apoptosis in living cells.