Mace Peter D, Riedl Stefan J, Salvesen Guy S
Biochemistry Department, University of Otago, Dunedin, New Zealand.
Program in Cell Death and Survival Networks, NCI Designated Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.
Methods Enzymol. 2014;544:161-78. doi: 10.1016/B978-0-12-417158-9.00007-8.
Apical caspases 8, 9, and 10 are only active as dimers. These dimers are unstable, and to characterize their activity they need to be maintained in vitro in a dimeric state. We provide updated methods for those looking to characterize various aspects of caspase function. We describe full methods for those looking to activate caspases in vitro using kosmotropic reagents, an essential step in characterizing upstream (apical) caspases. We detail methods for fusion of caspase domains to engineered dimerization domains as an alternative method to trigger regulated dimerization of caspases. We also describe methods to determine caspase activity profiles in cells and provide methods for studying the ability of SMAC-mimetic reagents to release inhibition of caspases by IAPs.
顶端半胱天冬酶8、9和10仅以二聚体形式具有活性。这些二聚体不稳定，为了表征它们的活性，需要在体外将它们维持在二聚体状态。我们为那些希望表征半胱天冬酶功能各个方面的人提供了更新的方法。我们描述了使用促离子型试剂在体外激活半胱天冬酶的完整方法，这是表征上游（顶端）半胱天冬酶的关键步骤。我们详细介绍了将半胱天冬酶结构域与工程化二聚化结构域融合的方法，作为触发半胱天冬酶调控二聚化的替代方法。我们还描述了确定细胞中半胱天冬酶活性谱的方法，并提供了研究SMAC模拟试剂释放IAP对半胱天冬酶抑制作用能力的方法。
