Bouchier-Hayes Lisa, Oberst Andrew, McStay Gavin P, Connell Samuel, Tait Stephen W G, Dillon Christopher P, Flanagan Jonathan M, Beere Helen M, Green Douglas R
Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
Mol Cell. 2009 Sep 24;35(6):830-40. doi: 10.1016/j.molcel.2009.07.023.
Caspase-2 is an initiator caspase activated in response to heat shock and other stressors that induce apoptosis. Activation of caspase-2 requires induced proximity resulting after recruitment to caspase-2 activation complexes such as the PIDDosome. We have adapted bimolecular fluorescence complementation (BiFC) to measure caspase-2 induced proximity in real time in single cells. Nonfluorescent fragments of the fluorescent protein Venus that can associate to reform the fluorescent complex were fused to caspase-2, allowing visualization and kinetic measurements of caspase-2 induced proximity after heat shock and other stresses. This revealed that the caspase-2 activation platform occurred in the cytosol and not in the nucleus in response to heat shock, DNA damage, cytoskeletal disruption, and other treatments. Activation, as measured by this approach, in response to heat shock was RAIDD dependent and upstream of mitochondrial outer-membrane permeabilization. Furthermore, we identify Hsp90alpha as a key negative regulator of heat shock-induced caspase-2 activation.
半胱天冬酶 -2是一种起始半胱天冬酶，在响应热休克和其他诱导细胞凋亡的应激源时被激活。半胱天冬酶 -2的激活需要在募集到半胱天冬酶 -2激活复合物（如PIDDosome）后产生诱导性接近。我们采用了双分子荧光互补（BiFC）技术来实时测量单细胞中半胱天冬酶 -2诱导的接近情况。荧光蛋白金星的非荧光片段可相互结合重新形成荧光复合物，将其与半胱天冬酶 -2融合，从而能够在热休克和其他应激后可视化并动态测量半胱天冬酶 -2诱导的接近情况。这表明，在响应热休克、DNA损伤、细胞骨架破坏及其他处理时，半胱天冬酶 -2激活平台出现在细胞质而非细胞核中。通过这种方法测量，响应热休克时的激活依赖于RAIDD，且在线粒体外膜通透性改变的上游。此外，我们确定热休克蛋白90α是热休克诱导的半胱天冬酶 -2激活的关键负调节因子。
