Nam Young-Jae, Mani Kartik, Wu Lily, Peng Chang-Fu, Calvert John W, Foo Roger S-Y, Krishnamurthy Barath, Miao Wenfeng, Ashton Anthony W, Lefer David J, Kitsis Richard N
Department of Medicine, Cardiovascular Research Center, and Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 2007 Feb 23;282(8):5522-8. doi: 10.1074/jbc.M609186200. Epub 2006 Dec 1.
Efficient induction of apoptosis requires not only the activation of death-promoting proteins but also the inactivation of inhibitors of cell death. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both central apoptosis pathways. Despite its potent inhibition of cell death, cells that express abundant ARC eventually succumb. A possible explanation is that ARC protein levels decrease dramatically in response to death stimuli. The mechanisms that mediate decreases in ARC protein levels during apoptosis and whether these decreases initiate the subsequent cell death are not known. Here we show that endogenous ARC protein levels decrease in response to death stimuli in a variety of cell contexts as well as in a model of myocardial ischemia-reperfusion in intact mice. Decreases in ARC protein levels are not explained by alterations in the abundance of ARC transcripts. Rather, pulse-chase experiments show that decreases in steady state ARC protein levels during apoptosis result from marked destabilization of ARC protein. ARC protein destabilization, in turn, is mediated by the ubiquitin-proteasomal pathway, as mutation of ARC ubiquitin acceptor residues stabilizes ARC protein and preserves its steady state levels during apoptosis. In addition, this degradation-resistant ARC mutant exhibits improved cytoprotection. We conclude that decreases in ARC protein levels in response to death stimuli are mediated by increased ARC protein degradation via the ubiquitin-proteasomal pathway. Moreover, these data demonstrate that decreases in ARC protein levels are a trigger, and not merely a consequence, of the ensuing cell death.
高效诱导细胞凋亡不仅需要促死亡蛋白的激活，还需要细胞死亡抑制剂的失活。ARC（含半胱天冬酶募集结构域的凋亡抑制因子）是一种内源性凋亡抑制剂，可拮抗两条主要的凋亡途径。尽管ARC对细胞死亡有强大的抑制作用，但表达大量ARC的细胞最终仍会死亡。一种可能的解释是，ARC蛋白水平会因死亡刺激而显著下降。目前尚不清楚在细胞凋亡过程中介导ARC蛋白水平下降的机制，以及这些下降是否引发随后的细胞死亡。在这里，我们表明，在多种细胞环境以及完整小鼠的心肌缺血再灌注模型中，内源性ARC蛋白水平会因死亡刺激而下降。ARC蛋白水平的下降不能用ARC转录本丰度的改变来解释。相反，脉冲追踪实验表明，细胞凋亡过程中ARC稳态蛋白水平的下降是由于ARC蛋白的显著不稳定所致。反过来，ARC蛋白的不稳定是由泛素-蛋白酶体途径介导的，因为ARC泛素受体残基的突变会使ARC蛋白稳定，并在细胞凋亡过程中保持其稳态水平。此外，这种抗降解的ARC突变体表现出更好的细胞保护作用。我们得出结论，死亡刺激导致的ARC蛋白水平下降是由泛素-蛋白酶体途径介导的ARC蛋白降解增加所致。此外，这些数据表明，ARC蛋白水平的下降是随后细胞死亡的触发因素，而不仅仅是其结果。
