Department of Biochemistry, University of Zurich, Switzerland.
Cell Death Differ. 2010 Apr;17(4):710-8. doi: 10.1038/cdd.2009.155. Epub 2009 Oct 23.
Caspases are the key players of apoptosis and inflammation. They are present in the cells as latent precursors, procaspases, and are activated upon an apoptotic or inflammatory stimulus. The activation mechanism of caspases has been studied extensively by biochemical and biophysical methods. Additional structural information on active caspases with a variety of different inhibitors bound at the active site is available. In this study, we investigated the cleavage mechanism of caspase-8 from its zymogen to active caspase-8 by solution NMR and by biochemical methods. The intermolecular cleavage reaction using the catalytically inactive C285A procaspase-8 mutant is triggered by adding caspase-8 and followed by (15)N,(1)H-NMR spectroscopy. The spectrum that initially resembles the one of procaspase-8 gradually over time changes to that of caspase-8, and disappearing peaks display exponential decaying intensities. Removal of either one of the cleavage recognition motifs in the linker, or phosphorylation at Tyr380, is shown to reduce the rate of the cleavage reaction. The data suggest that dimerization repositions the linker to become suitable for intermolecular processing by the associated protomer. Furthermore, analysis of inhibitor binding to the active caspase-8 reveals an induced-fit mechanism for substrate binding.
半胱天冬酶是细胞凋亡和炎症的关键执行者。它们以潜伏前体，即 procaspases 的形式存在于细胞中，并在凋亡或炎症刺激下被激活。半胱天冬酶的激活机制已通过生化和生物物理方法进行了广泛研究。此外，还提供了与各种不同抑制剂结合在活性部位的活性半胱天冬酶的附加结构信息。在这项研究中，我们通过溶液 NMR 和生化方法研究了 caspase-8 从酶原到活性 caspase-8 的切割机制。使用催化失活的 C285A 原 Caspase-8 突变体进行的分子间切割反应通过添加 Caspase-8 触发，并通过 (15)N，(1)H-NMR 光谱进行跟踪。最初类似于原 Caspase-8 的光谱随时间逐渐变化为 Caspase-8 的光谱，消失的峰显示出指数衰减强度。在连接体中去除任一切割识别基序，或 Tyr380 磷酸化，均显示出降低切割反应速率的作用。数据表明，二聚化使连接体重新定位，以便与其相关的前体分子间进行加工。此外，对抑制剂与活性 Caspase-8 结合的分析揭示了底物结合的诱导契合机制。
