Determining the contributions of caspase-2, caspase-8 and effector caspases to intracellular VDVADase activities during apoptosis initiation and execution
详细信息 查看全文
- 作者：M. Eugenia Delgado ; Magnus Olsson ; Frank A. Lincoln ; Boris Zhivotovsky ; Markus Rehm
- 关键词：Apoptosis ; Fö ; rster Resonance Energy Transfer (FRET) ; Caspase-2 ; Caspase-8 ; Single cell analysis
- 刊名：Biochimica et Biophysica Acta (BBA)/Molecular Cell Research
- 出版年：2013
- 出版时间：October, 2013
- 年：2013
- 卷：1833
- 期：10
- 页码：2279-2292
- 全文大小：1261 K
文摘
Apoptosis signaling crucially depends on caspase activities. Caspase-2 shares features of both initiator and effector caspases. Opinions are divided on whether caspase-2 activity is established during apoptosis initiation or execution in response to DNA damage, death receptor stimulation, or heat shock. So far, approaches towards measuring caspase-2 activity were restricted to analyses in cell homogenates and extracts, yielded inconsistent results, and were often limited in sensitivity, thereby contributing to controversies surrounding the role of caspase-2 during apoptosis. Furthermore, caspases overlap in substrate specificities, and caspase-8 as well as effector caspases may cleave the optimal VDVAD recognition motif as well. We therefore generated a highly sensitive F?rster resonance energy transfer (FRET) substrate to determine the relative contribution of these caspases to VDVADase activity non-invasively inside living cells. We observed limited proteolysis of the substrate during apoptosis initiation in response to death receptor stimulation by FasL, TNF¦Á and TRAIL. However, this activity was attributable to caspase-8 rather than caspase-2. Likewise, no caspase-2-specific activity was detected during apoptosis initiation in response to genotoxic stress (cisplatin, 5-FU), microtubule destabilization (vincristine), or heat shock. The contribution of caspase-2 to proteolytic activities during apoptosis execution was insignificant. Since even residual, ectopically introduced caspase-2 activity could readily be detected inside living cells in our measurements, we conclude, in contrast to several previous studies, that caspase-2 activity does not contribute to apoptosis in the scenarios investigated, and that instead caspase-8 and effector caspases are the most significant VDVADases during canonical apoptosis signaling.