Rodriguez Diego A, Green Douglas R
Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA.
Methods Mol Biol. 2018;1857:71-83. doi: 10.1007/978-1-4939-8754-2_7.
Necroptosis, a form of regulated necrosis, is triggered by a variety of signals that converge to activate receptor interacting protein kinase-3 (RIPK3), consequently promoting the direct phosphorylation and activation of the mixed lineage kinase like (MLKL) protein. Active MLKL executes necroptosis by disrupting the integrity of the plasma membrane. Stimuli that can induce necroptosis include ligation of death receptors (a subset of the TNFR family), toll-like receptors (in particular, TLR3 and TLR4), interferons, and the intracellular viral sensor, DAI/ZBP1, among others. To study the process in more detail, it is useful to have a means to directly activate RIPK3. Here we provide protocols and procedures to artificially induce necroptotic cell death by drug-induced forced dimerization of RIPK3. We also provide information on specific kinase inhibitors, procedures to monitor RIPK3 and MLKL activation, and real-time quantification of cell death.
坏死性凋亡是一种程序性坏死形式，由多种信号触发，这些信号汇聚以激活受体相互作用蛋白激酶-3（RIPK3），从而促进混合谱系激酶样（MLKL）蛋白的直接磷酸化和激活。活化的MLKL通过破坏质膜的完整性来执行坏死性凋亡。可诱导坏死性凋亡的刺激包括死亡受体（肿瘤坏死因子受体家族的一个子集）的连接、Toll样受体（特别是TLR3和TLR4）、干扰素以及细胞内病毒传感器DAI/ZBP1等。为了更详细地研究这一过程，有一种直接激活RIPK3的方法很有用。在这里，我们提供了通过药物诱导RIPK3强制二聚化来人工诱导坏死性凋亡细胞死亡的方案和程序。我们还提供了关于特定激酶抑制剂的信息、监测RIPK3和MLKL激活的程序以及细胞死亡的实时定量方法。
